ADIOLOGY 
11111 

NCOLOGY 
March 1999 Vol. 33 No. 1 Ljubljana 
ISSN 1318-2099 



.TI. 




Editorial office Radiologij and OncologtJ March 1999 Institute oj Oncology Vol. 33 No. 1 Vrazov trg 4 Pages 1-85 SI-1000 Ljubljana ISSN 1318-2099 Slovenia UDC 616-006 Phone: +386 611320 068 CODEN: RONCEM Phone/Fax: +386 61 1337 410 E-mail: gsersa@onko-i.si 
Aims and scope 

Radiology and Oncology is a journal devoted to publication oj original contributions in diagnostic and interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radia/ion protection. 
Editor-in-Chief  Editor-in-Chief Emeritus  
GregorSerša  Tomaž Benulic  
Ljubljana, Slovenia  Ljubljana, Slovenia  
Executive Editor  Editor  
Viljem Kovac  Uroš Smrdel  
Ljubljana, Slovenia  Ljubljana, Slovenia  

Editorial board 

Marija Auersperg Be?aFornet MajaOsmak 
Ljubljana, Slovenia Budapest, Hungary Zagreb, Croatia 
Nada Bešenski Tullio Giraldi Branko Palcic 
Zagreb, Croatia Trieste, Italy Vancouve1; Canada 
Karl H. Bohuslavizki Andrija Hebrang JuricaPapa 
Hamburg, Gerrnany Zagreb, Croatia Zagreb, Croatia 
HarisBoko Ltiszl6 Horvtith Dušan Pavcnik 
Zagreb, Croatia Pecs, Hungary Portland, USA 
Nataša V. Budihna Berta Jereb Stojan Plesnicar 
Ljubljana, Slovenia Ljubljana, Slovenia Ljubljana, Slovenia 
Marjan Budilma Vladimir Jevtic Ervin B. Podgoršak 
Ljubljana, Slovenia Ljubljana, Slovenia Montreal, Canada 
Malte Clausen H. Dieter Kogelnik Jan C. Roos 
Hamburg, Germany Salzburg, Austria Amsterdam, Netherlands 
Christoph Clemm Jurij Lindtner Slavko Šimunic 
Miinchen, Germany Ljubljana, Slovenia Zagreb. Croatia 
Mario Co1·si Ivan Lovasic Lojze Smid 
Udine, Italy Rijeka, Croatia Ljubljm!_a,Slovenia 
Christian Dittrich Marijan Lovrencic Borut Stabuc 
Vienna, Austria Zagreb, Croatia Ljubljana, Slovenia 
Ivan Drinkovic LukaMilas Andrea Veronesi 
Zagreb, Croatia Houston, USA Aviano, Italy 
Gillian Duchesne Metka Milcinski Živa Zupancic 
Melbourne, Australia Ljubljana, Slovenia Ljubljana, Slovenia 
Publishers 

Slovenian Medica/ Association -Slovenian Association oj Radiology, Nuclear Medicine Society, Slovenian Society far Radiotherapy and Oncology, and Slovenian Cancer Society Croatian Medica/ Association -Croatian Society oj Radiology 
Affiliated with 
Societas Radiologorum Hungarorum Friuli-Venezia Giulia regional groups oj S.I.R.M. (Italian Society oj Medica/ Radiology) 

Copyright © Radiology and Oncology. Ali rights reserved. 
Reader for English 
Olga Shrestha Mojca Cakš 
Keywords 
Eva Klemencic 
Secretaries 
Milica Harisch 

Betka Savski 
Design 


Monika Fink-Serša 
Printed by 
Imprint d.o.o., Ljubljana, Slovenia 
Published quarterly in 750 copies 
Bank account number 50101 679 901608 
Foreign currency account number 
50100-62 0-133-2 762 0-5130/ 6 
NLB -Ljubljanska banka d.d. -Ljubljana 

Subscription fee far institutions $ 100 (16000 SIT), individuals $ 50 (5000 SIT) 
The publication oj this journal is subsidized by the Ministry oj Science and Technology oj the Republic oj Slovenia. 
According to the opinion oj the Govermnent oj the Republic oj Slovenia, Public Relation and Media Office. The journal Radiology and Oncology is a publication oj infonnative value, and as such subject to taxation by 5 % sales lax. 
Indexed and abstracted by: 
BIOMEDICINA SLOVENICA 
CHEMICAL ABSTRACTS 
EMBASE / Excerpta Medica 
This journal is prin ted on acid-free paper 

Radiology and Oncology is available on lhe internet at: http://www.onko-i.si/radiolog/rno.htm 

ADIOLOGY 
AND 


NCOLOGY 


Ljubljana March 1999 Vol. 33 No. 1 
CONTENTS 
RADIOLOGY ANO COMPUTERIZED TOMOGRAPHY 


.TI. 
ISSN 1318-2099 UDC 616-006 CODEN: RONCEM 

Pharmaco-mechanical technique for selective thrombolysis in peripheral vessels Hadjiev ], Horvdth L, Mezi5fi B, Szalay G  1  
Spectral characteristics of the high-resolution liver CT data: detection of iron-overload and cirrhosis Kurbel S, Kristek B, Kovacic D, Glavina K, Kurbel B  9  
NUCLEAR MEDICINE  
Standardized image documentation in nuclear medicine Bohuslaviz/ci KH, Buchert R, Mester ], Clause11 M  15  
Detection of lymph node metastasis from osteosarcoma with 99mTc-MDP scintigraphy. Case report Ilic D, Zafirovs/ci G, Simova N, Zisovski N, Glamocanin S, Janevs/ca V, Tolevs/ca C, Vas/cova O, Smnardzis/ci M  23  
EXPERIMENTAL ONCOLOGY  

A correlation of NK cytolitic test and BLT esterase test in determining activity of NK cells, stimulated by tumor target cells 
Ihan A 
Micronuclei in cytokinesis-blocked lymphocytes of patients following iodine-131 radiotherapy 
Kašuba V, Kusic Z, Graj-Vrhovac V 
Prognostic relevance of urokinase plasminogen activator and its inhibitors in patients with breast cancer 
Borštnar S, Cufer T, Vrhovec I, Rudolf Z 
CLINICAL ONCOLOGY 
Radiotherapy for choroidal neovascularization of age-related macular degeneration Wagner W, Beeke E, Barsnick P  55  
Second malignancy after radiotherapy for seminoma Fuchshuber PR, Lee R], McGrath B, Gibbs ]F, Kraybill WG, Proulx GM  59  
Sarcomas of the bladder: A case report with review of the literature Proulx GM, Gibbs ]F, Lee R], Velagapudi S, Huben R  63  
RADIOPHYSICS  
Epithermal neutron beam for BNCT in the IJS TRIGA reactor -modelling and experimental verification Maucec M, Glumac B, Rant ], Krištof E  69  
SLOVENIAN ABSTRACTS  77  
NOTICES  83  

Radiology and Oncology is covered in Biomedicina Slovenica, Chemical Abstract, and EMBASE / Excerpta Medica 
Radio/ Onco/ 1999; 33(1): 1-8. 
Pharmaco-mechanical technique for selective thrombolysis in peripheral vessels 
Janaki Hadjiev, Laszlo Horvath, Beata Mezofi, Gabriella Szalay 
Department of Radiology, Medica/ University Pecs, Hungary 
Purpose. The thrombus destroying power oj selective low-dose jibrinolytic treatment was intended to be amplijied with the mechanical Jarce oj the pulse-spray injector. The treatment time was expected to be shortened and the incidence oj complications to be reduced. Methods. Seventeen patients with arterial occlusion on the lower extremities underwent selective pulse­spray thrombolysis. Ali were treated according to a standard thrombolytic protocol. Streptokinase (Kabiki­nase®) 5.000-20.000 IU/h, or urokinase (Ukidan®) 20.000-40.000 IU/h and heparin 500-1.000 IU/h in physiologica/ saline were administered with pulse-spray injector (Angiodynamics®) til/ the total dissolving oj the thrombus. Results. Between 1995-1997, 17 patients were successjully treated with an average treatment tirne oj 12 hours,. No major complication occurred during this period. Surgical operation in the first 6 months ajter the treafment was needed in two cases because oj reappearance oj the clinica/ symptoms. Conclusion. The pharmacomechanical se/ective thrombolysis with the pulse-spray technique is a reliab/e, relatively rapid and saje method jor recanalization oj occluded native arteries. 
Key words: arterial occlusive diseases-drug therapy; thrombolytic therapy-methods 
Introduction 

The percutaneous intraarterial selective thrombolysis is a radiologically controlled minimal invasive therapy used mainly in the cases of arterial occlusions of the extremities. Since 1959, when a research group under the leadership of Fletcher introduced for the first tirne the fibrinolytic treatment with streptoki-
Received 23 Maj 1998 
Accepted 22 August 1998 
Correspondence to: Janaki Hadjiev MD, Departrnent of Radiology POTE, Ifjusag U. 13, H-7624 Pecs Hun­gary. Tei: +36 72 324 122; Fax: +36 72 311 214. 
nase, a great progress has been achieved in 
2 

this field.1, 
The method of drug activation of the fibri­nolytic system through an intraarterial catheter first applied by Dotter in 1974 has been routinely used in the radiologic depart­ments since the middle of the 1980's. In 1989, Bookstein and his co-workers intro­duced the pulse-spray pharmacomechanical treatment method.3 The thrombus removing power of the selective low-dose fibrinolytic treatment was boosted by the mechanical force of a pulse-spray injector incredibly increasing the effective surface of drug and dot interaction. 
I-ladjiev J et ni./ Tromholysis in peripheral vesse/s 
After an appropriate selection, patients with lower extremity arterial occlusion have been undergoing selective intraarterial thrombolysis with pulse-spray infusion in our angiographic department since the end of 1995. 


Material and methods 
Patient selection: For the performance of selective thrombolysis there are various indi­cations and contraindications (Table 1). Appropriate selection and monitoring of the patients are of significant importance for a successful treatment. According to etiology, occlusions may be classified in the following three groups; -arterial embolization-usually from the 
left side of dilated heart, after cardiac arrhythmic attack due to an aortic aneurism. Patients were usually admitted in the hospital in an acute stage of clinical 
manifestation,  1-2  hours  after  the  
embolization;  
-arterial  thrombembolization  without  a  

preexisting arterial disease -mainly of iatrogenic ongm, usually with good response to thrombolytic treatment, solved within a few hours; -arterial thrombembolization following a 
preexisting vessel disease -the most fre­
quently presented form of disease in the 
lower extremities. 
An intimal damage caused by the diabetic or atherosclerotic disease leads to an increase of the surface contact activity and, by desta­bilizing the homeostasis, activates the chain reaction of coagulation. All the patients who underwent selective thrombolysis in our department were classified into this group (Table 2). They had acute or subacute clinical signs of severe limb ischemia with a preced­ing history of claudication. All the patients with proper indication for pulse-spray selec­tive thrombolysis were treated in our angio-
Radiol Oncol 1999; 33(1): 1-8. 
Table 2. Patients and results in selective pulse-spray thrombolysis 
Patients 17 male/female 10/7 Average age 59 (39-83) Main risk factors AS* 12 
DM* 
5 Fontaine stage III 
7 
stage IV 10 Therapeutic succes 17 Roecclusion within 6 monts 2 
AS* -Atherosclerosis obliterans; DM'· -Diabetes mellitus 
Table 3. Treatment protocol in selective pulse-spray thrombolysis 
l. Baseline angiogram 
2. 
Introduction of guide wire 

3. 
Infusion catheter selection 

4. 
Thrombolytic agents infusion 

5. 
Control angiography 

6. 
Catheter removal 

7. 
Postprocedure management, aftercare 


graphic department. No one of the patients was excluded from this study. Seventeen patients underwent pulse-spray thrombolysis of acute arterial occlusion which was caused by atherosclerosis obliterans and by diabetic angiopathy in 12 and in 5 cases, respectively. 


Catheterization technique 
In our angiographic department, a well-tested treatment protocol was used (Table 3). The main steps of the treatment protocol are schematically presented in Figure 1; -pulse-spray technique -the jet spray of the 
lytic infusion causes a significant change in the intrathrombotic microstructure and improves the effectiveness of the method enlarging the surface for the action of the urokinase or streptokinase (Figure 2). The pulse-spray infusion was performed with an infusion pump through the catheter without blocking the end hole. 

Hadjiev J et ni. / Trombolysis in peripheral vessels 
Table l. Indications and contraindications for selective pulse-spray thrombolysis 
Indications 
acute and chronic native artery occlusion acute and chronic graft occlusion reoclusion after percutan angioplasty 
Contraindications 

Absolute 

Active interna! bleeding Cerebrovascular accident, disease or surgical intervention within the last 2 months 
Relative Surgical operation or parenchymal organ biopsy within the last 2 weeks Recent major trauma Uncontrollable hypertension Gastrointestinal ulcer 
Pregnancy/postpartum 
Embolization from a distant source 


infused lytic drugs -in arterial selective thrombolysis, the drug dose administered with the pulse-spray technique was lower than the ones indicated in systemic infu­sion protocols. The reports of early clinical successes in literature show significant 
9 
preference of urokinase to streptokinase.4­However, this observation has not been proved yet although it was explained in randomized trials with pharmacokinetic mechanisms of the drugs.10 In our intraar­terial treatments, 20000-40000 IU/hour urokinase (Ukidan®), or 5000-20000 IU/ hour streptokinase (Kabikinase®) and 500-1000 IU/hour heparin were adminis­trated through the catheter. 

Postprocedure management: In order to prolong the patency rate of the reopened ves­sels, a post-procedural care and change in lifestyle was proposed to all the patients. To prevent puncture site bleeding, false pulsat­ing aneurysms and extensive haematoma 20­30 minutes after manual compression, a sand sack was placed on the bandage of the punc­ture site for 6 hours. Care was taken that the patients made exercises to increase blood flow in the affected extremity in order to pre­vent local or venous thrombosis due to the transient stasis under the compression and slow flow because of bed rest. The patients were kept in bed for the next 24 hours. Diuretics, if any, were excluded from the treatment in the ward, and, in cases of dia­betes mellitus, the concentration of serum glucose was kept steadily within the normal limits. The 3rd-5th day after the treatment a daily dose of 2 x 50 to 2 x 100 mg of sodium pentosan polysulphate (SP 54®) was admin­istered i.v. (occasionally s.c.). Smoking was prohibited to the patients while life-long physical exercises, low-fat and low-choles­terol diet were recommended to them. Con­trol examinations (physical and Doppler­flowmonitoring) and laboratory tests (hemat­ocrite, serum cholesterol, serum triglycerid, plasma fibrinogen) were made at the dis­charge from the hospital, 30 days and 3 months after the therapy and, later on, in 6 month intervals for life tirne. 


Results 

Selective thrombolysis was considered clini­cally successful in the cases in which the ischemic signs disappeared or significantly improved for a period of more than 30 days 
Radio/ 011co/ 1999; 33(1): 1-8. 

Hadjiev J et a/. / Trombolysis in periphera/ vesse/s 

1/a 

Figure l. Main steps of treatment procedure. After the baseline angiogram a guide wire is introduced into the thrombus (I/a). If this is not possible the treatment starts with continuous selective thrombolytic infusion (I/b). If possible the infusion catheter is positoned in the throm­bus leaving a thrombus plug at the <listal end. After excluding the chance of accidental subintimal position pulse-spray lysis is initiated (II). After 4-6 hours of treat­ment if the control angiography shows reopening of the vessel (III), the pulse-spray technique may be converted to continuous infusion (IV) till the total dissolving of the thrombus. In cases of small persistent wall irregularities 
(V) percutaneous transluminal angioplasty is also per­formed. 
after the therapy. In ali 17 patients, the occluded arteries of the lower extremity were successfully reopened. In two of the treated cases, a surgical treatment was performed within the first 6 month after the therapy because of the recurrence of clinical symp­toms. The first patient had diabetes mellitus for more than 15 years. After successful lytic treatment of the occlusion in the <listal part of the popliteal artery, an amputation of the third left foot finger (previous amputation of the 4th and 5th finger on the same side in his clinical history) was carried out. The other patient did not adhere to the proposed post-procedure drug therapy and lifestyle changes. He underwent a successful translu­minal endarterectomy of the popliteal artery 4 months after our treatment. Occult bleed­ing, allergic reactions, myocardial spasm, or <listal embolization with an occurrence rate of 0.5-5%, which were often reported in litera­ture, 4·11-15 were not observed during our pro­cedures in spite of the careful control with laboratory tests, angiography and instrumen­ta! flow measurements. 
Discussion 

After the thrombolytic treatment we per­formed, in three of our cases, a percutaneous transluminal angioplasty on the chronic ste­notic plaques detected by the control angio­graphy and made visible only after solving the thrombotic deposits from the surface of the vascular wall. 
To our opinion, a continuous selective infusion cannot provide an optimal concen­tration of the lytic enzymes in the thrombus where the activation of the plasminogen is required. The pharmacomechanical throm­bolysis, which has appeared after a vast number of experimental research trials includes, -shooting tiny jets of thrombolytic agent 
into the mass of thrombotic deposits; -maceration of the thrombus; -supplying in this way a larger surface for 
the action of the lytic enzyme, -providing a simultaneous destruction of 
the majority of fibrin in the treated seg­
ment of the occlusion. 
The maceration of thrombembolism is due to the jet effect of the infusion, i.e. its mechanical power. The duration of this process and the total thrombolysis as report­ed in literature has really a broad sca­
·13·14·16·17 
le.4•5The explanation of this fact seems to be found in a multifactorial reason 

Radiol Oncol 1999; 33(1): 1-8. 
Hadjiev J et al. / Trombolysis in peripheral vessels s 

Figure 2. The pulse-spray system. Using the mechanical force of an automatic pump on the 1 cc syringe a bolus (0,1-0,5 ml) of saline solution containing 10% thrombolytic agents is infused in the thrombus with high pressure through the pressure responsive slit orifices of the catheter. The frequency of the jet spray varies from 6 to 120 seconds. 
theory. The average treatment time in our study was 12 hours and was calculated from the treatment times ranging from 6 to 27 hours. These differences were mainly due to the site and size of the thrombus, its underly­ing disease and duration of ischemic condi­tions. It has been observed that chronic limb ischemia seems to be more resistant to thrombolysis, 18 but it should not considered as a relative contraindication.13J4 
After an appropriate selection of patient, premedication and catheter technique, the next important step to achieve a really good and long-term clinical success is the estab­lishment of a proper thrombolytic treatment plan and the monitoring of the patients. The role of partial thrombin time (PTT), thrombin time (TT) and fibrinogen serum concentra­tion in „holding the treatment in hands" is discussed in the literature, and fibrinogen level under 1 g/1 is often associated with 
n 14 17 

occult bleeding.1, 10,, , This is especially significant in patients with clinical history of ulcer, cerebrovascular accident, recent major trauma, or surgery, especially around the pancreas and the prostate gland. 
With the use of the pulse-spray technique, new requirements emerged. In order to reopen the occluded vessel as quickly as pos­sible often thrombolysis of the whole length of the thrombus was attempted from the 
20 

very start of the procedure.19, An occasion­al distal embolization should be further treat­ed with thrombolytic infusion or percuta­neous thrombus aspiration. Other teams start the lytic therapy from the distal end of the thrombus and go further in proximal direction.21 In the majority of our cases, most of these techniques proved to be non benefi­cial because of the hardness of the occlusion. Even in those cases, when the guide wire was easily introduced into the thrombus we sug­gest that, because of thehigh pressure, a short thrombotic plug left behind at the dis-
Radiol Oncol 1999; 33(1): 1-8. 

Hadjiev J et al. / Trombolysis in peripheral vessels 

Radio/ Oncol 1999; 33(1): 1-8. 

Hadjiev J et ni. / Trombolysis in peripheral vesse/s 


Figure 3 a-d. Case rcport. E. K. 63 year old female patient with a clinical history of diabetes mellitus for 10 years, compli­cated by hypertension in the last 3.5 years. Trigliceride and cholesterol level were slightly elevated. Complains started a week before her arrival in the department and increased in the last 2 days. The clinical picture was Fontaine IV. The right leg under the knee joint was livid, cyanotic and thcre was no pulse in the popliteal artery and distaly to that. 
Selective angiography was performed with a cross-over technique, which demonstratcd a total occlusion of the right popliteal artery (a, b). Pulse-spray infusion was initiated after positioning the catheter in the thrombus. The result of 1 hour control angiography was encouraging, so, pulse-spray technique was continued for further four hours. After nine hours of treatment, the control angiography showed total rccanalization (c, d). The clinical picture at discharge from the hospital was Fontaine 1-II. Three years after the procedure, strictly adhering to the postprocedure protocol, the patient 
had no significant increase of ischaemic symptoms. 
tal end might be useful for the profilaxis of embolization (Figure 3). 
The patency rate of the reopened arteries is in strong dependence of a proper postpro­cedural management. It seems to be obvious that, without a proper aftercare, thrombolysis is not going to result in a satisfactory long­term patency, especially in cases of pre-exist­ing progressive disease, although this assumption has not been proved in random­ized studies on selective thrombolysis. 
Conclusions 
Till date, there has been a relative reluctance from the wide use of selective thrombolysis, mainly because of the long duration of the procedure, quite high radiation, Jack of expe­rience and a number of other reasons. The introduction of the pharmacomechanical method proved to be a great step forward in managing the occlusion with combined chemical and physical forces. The mechanical power of the jet spray multiplies the action of the lytic drug helping, on the one hand, its deeper penetration into the thrombus and, on the other, mechanically destroying the thrombus. 
The selective thrombolysis with pulse­spray technique is a radiological treatment of occlusions which may be part of the complex treatment, including surgical procedures, or applied as a curative therapy alone. 
With an appropriate selection of patients, a well-tested treatment protocol and proper posHreatment care of the patients both, the acute and subacute artery occlusions may be successfully treated and amputation rate sig­nificantly lowered. 

References 
1. 
Fletcher AP, Sherry S, Alkjaersig N. The mainte­nance of a sustained thrombolytic state in man. II. Clinical observations on patients with myocardial infarction and other thrombolytic disorders. J Ciin I11vcst 1959; 38: 111'l -34. 

2. 
Meyer JS. Therapeutic thrombolysis in cerebral thromboembolism Double-bind evaluation of intravenous plasmin therapy in carotid and midle cerebral artery occlusion. Neurology 1963; 13: 972­83. 

3. 
Bookstein JJ, Fellmeth B, Roberts A, Davis G. Pulsed-spray pharmacomechanical thrombolysis: preliminary clinical results. AJR 1989; 152: 1097-9. 

4. 
Belkin M, Belkin B, Bucknam CA. Intrarterial fib­rinolytic therapy: efficacy of streptokinase vs. urokinase. Are/z Surg 1986; 121: 769-75. 

5. 
Groar RA, Olin J, Bartholomew JR. Efficacy and safety of intraarterial local infusion of streptoki­nase, urokinase, or tissue plasminogcn activator for peripheral arterial occlusion: a retrospective review. J Vase Med Biol 1990; 2: 310-15. 

6. 
Janosik JE, Bcttmann MA, Kaul AF, Sonney PF. Therapeutic alternativcs for subacute peripheral arterial occlusion: comparison by outcome, length of stay and hospital charges. lnves/ Radio! 1991; 26: 921-27. 

7. 
Traughber PB, Cook PS, Michlos TJ, Miller FJ. Intraarterial fibrinolytic therapy for popliteal and tibial artcry obstruction: comparison of streptoki­nase to urokinase. AJR 1987; 149: 543-9. 

8. 
Van Breda A, Groar RA, Katzen BT. Relative cost effectiveness of urokinase versus streptokinase in the treatment of peripheral vascular disease. ]VIR 1991; 2: 77 -86. 


Radio/ Oncol 1999; 33(1): 1-8. 

Hadjiev J et a/. / Trombolysis in periphera/ vessels 
9. 
Van Breda A, Katzen BT, Deutsch AS. Urokinase versus streptokinase in local thrombolysis. Radiol­ogy 1987; 165: 109-13. 

10. 
Holden RW. Plasminogen activators: pharmacolo­gy and therapy. Radiology 1990; 174: 993-1006. 

11. 
Gardiner GA, Sullivan KL. Complications of regional thrombolytic therapy. Currenl practice oj interventiona/ radiology. Philadelphia: BC Decker; 1991. p. 87-96. 

12. 
Hess H, Mietaschk A, Briickl R. Peripheral arterial occlusions: a 6-year experience with local low­dose thrombolytic therapy. Radiology 1987; 163: 753-61. 

13. 
Kandarpa K. Peripheral arterial thrombolysis: state of the art. 2nd Interventional Workshop on Interventional Radiology. Circulation 1995; 1: 14­15. 

14. 
Kandarpa K, Chopra PS. Intraarterial thromboly­sis of lower extremity occlusion a prospective, randomized comparison of forced periodic infu­sion and conventional slow continuous infusion. Radiology 1993;188: 861-70. 

15. 
Sullivan KL, Gardiner GL, Sharpiro MJ. Accelera­tion of thrombolysis with high-dose transthrom­bus bolus technique. Radiology 1989; 173: 805-12. 


16. 
Eisenbud DE, Brener BJ, Schoenfeld R. Treatment of acute vascular occlusions with intra-arterial urokinase. Am] Surg 1990; 160: 160-7. 

17. 
McNamara TO, Bomberger RA. Factors affecting initial and six month patency rates after intraarte­rial thrombolysis with high dose urokinase. Am J Surg 1986; 152: 709-15. 

18. 
Luppatelli L, Barzi F, Corneli P. Selective throm­bolysis with low-dose urokinase in chronic arte­riosclerotic occlusions. Cardiovasc Intervent Radio/ 1988; 18: 123-30. 

19. 
Motarjeme A. Thrombolytic therapy in arterial occlusion and graft thrombosis. Seminars in Vascu­lar Surgery 1989; 2: 155-65. 

20. 
Valji K, Bookstain J, Roberts A, Davis G. Pharma­co mechanical thrombolysis and angioplasty in the management of clotted hemodialysis grafts: early and late clinical results. Radiology.1991; 178: 243-9. 

21. 
Mevissen MW, Monor PL. Symptomatic native arterial and graft occlusions: early experience with „over-the-wire" thrombolysis. ]VIR 1990; 1: 43-9. 





Radio/ Oncol 1999; 33(1): 1-8. 
Radio/ Oncol 1999; 33(1): 9-14. 
Spectral characteristics of the high-resolution liver CT data: 
detection of iron-overload and cirrhosis 
Sven Kurbel1 , Branka Kristek2 , Damir Kovacic3 , Krešimir Glavina2, Beatrica Kurbel4 
1 Dept. oj Oncology, 2 Dept. oj Radiology, 3 Clinic oj Surgery oj Osijek Clinical Ho spital & Clinic oj Surgery, 4Dept. oj Anaesthesiology University Hospital "Rebro", Zagreb, Croatia 
Background. Statistical methods and Fourier analysis oj CT values were used to detect the presence oj /iver cirrhosis and oj iron-overload in the sets oj high-resolution /iver CT data. Subjects and methods. Eleven /iver cirrhosis patients, seven hemodialysed patients with iron-overload and 51 control individuals were included. CT unit SIEMENS SOMATOM DRH was used (kernel 2, 0.2 mm wide and 2 mm thick pixels). Square sample areas, 50 pixels wide, two per control and three per patient were col/ected. The areas were statistically analysed and decomposed in 100 linear fragments (50 lines and 50 columns oj 50 pixels) far Fourier analysis. The mean and standard deviations oj harmonic powers were calculated far each sample. Results. The high-resolution CT data in the iron-overload patients differed from the controls by the increased mean density (:2.79 HU) and reduced power oj many harmonics (p<0.01). The high-resolution CT data oj the cirrhotic patients showed the decreased mean density (<50 HU) and increased power oj har­monics from 0.1 to 0.9 cycles/111111 (wavelengths from 10 to 1.1 mm) (p<0.01). Conclusions. Information extractable from the high-resolution /iver CT data can distinguish between the normal and cirrhotic, ar iron-overload livers. 


Key words: /iver cirrhosis, /iver diseases; iron overload; tomography, x-ray computed, Fourier analysis 
Introduction 
Ultrasound, CT and MR are dominant methods of liver imaging, mainly used for the detection of focal !iver diseases. Conventional CT images with large pixels are unable to define CT charac-
Received 20 July 1998 
Accepted 4 December 1998 Correspondence to: Sven Kurbel MD, PhD, Dept. of Oncology, Osijek Clinical Hospital, 31000 Osijek, Croatia; Tel.: (+385) 31 51 14 85; Fax: (+385) 31 51 22 22; E-mail: sven.kurbel@public.srce.hr 
teristics of the cirrhotic !iver. Fibrosis and altered !iver architecture, as the main pathologi­cal characteristics of !iver cirrhosis, do not sub­stantially alter the mean density on these images1 , presumably due to the effect of partial volume. Each voxel of the conventional abdomi­nal CT image contains parts of severa! !iver Iob­ules and the effect of partial volume thus dis­ables the visualisation of detailed densitometric texture. 
The present study was aimed to test whether the high-resolution CT values can distinguish 

Kur/Jel Set ni./ Deteclio11 oj iron-over/ond n11d cirrhosis wi//1 CT 
normal !iver images from the images of the cir­rhotic or iron-overload livers. Statistical meth­ods and Fourier analysis of CT values were used for this purpose. 
Patients and methods 

The control group consisted of 51 patients scheduled for abdominal CT examinations of kidneys, spine or other organs, without history or clinical signs of diffuse !iver diseases. Eleven patients with a clinic diagnosis of !iver cirrhosis were CT examined to exclude the possibility of !iver neoplasm. All patients were in the condi­tion of decompensated !iver cirrhosis with ascitic fluid on this or any of the previous hospi­tal stays. The iron-overload group consisted of seven hemodialysed patients with chronic renal failure and iron-overload (serum ferritin more than four times above normal). These patients were scheduled for the abdominal CT because of their renal or spine pathology. 
A single, 2 mm thick !iver CT image was taken through the central part of the !iver on a SIEMENS SOMATOM DR-H CT unit before the contrast application. We used the abdominal kernel 2, 720 projections in four seconds with zoom factor 5.2. Pixels were 0.2 mm wide and 2 mm thick in all images. 
CT data were stored and square areas wide 50 pixels (2500 pixels per square) were later transferred to a portable PC. A simple RS-232 adapter and XTALK program were used to con­nect the PC in the place of a DIGITAL control console. We used the command ABS/DIA/LIS from the measuring program of SOMATOM DRH to export the chosen area of CT values to the capture file on a portable PC. Files were edited in a DOS editor to discard textual infor­mation and, after that, the data were realigned to reconstruct the initial CT matrix. 
Fram these data simple statistical measures were calculated.2 Then, each sample area of CT values was decomposed in 50 columns and 50 rows (in total 100 linear segments of 50 pixels). 
These segments were analysed by the conven­tional Fourier analysis. Amplitudes of sine and cosine components were calculated for the har­monics ranging from O. to 25. (from O to 2.5 cycles/mm). Amplitudes of the cosine and the sine components were used to calculate the power of each harmonic in the linear segment.3 The mean harmonic power spectrum with stan­dard deviations was calculated for each sample. 
Observed parameters were tested by the cal­culation of ROC curves.4 Numbers of true posi­tive/negative and false positive/negative were used in 2·2 tables for the calculation of X2 val­ues.2 
Results 

Table 1 shows the statistical characteristics of the collected sets of CT data and calculated sets of contrast values. 
The high-resolution (HR) CT <lata of the liv­ers with iron-overload showed the increased mean density in comparison to the control sam­ples with the decreased mean densitometric contrast value. Both characteristics are probably caused by the abundant and evenly distributed iron in !iver tissue. 
On the contrary, the HR CT <lata of the cir­rhotic livers showed the reduced mean density and increased mean contrast value. 
Table 2 shows the results of Fourier analysis. Mean powers with a standard deviation are shown only for the first 10 harmonics. Omitted harmonics include the O. hannonic, whose power equals the mean densities already shown in Table 1. The powers of higher harmonics (from 11. to 25.) were similar in the controls and groups of patients and considered unimportant. 
In comparison to the HR control CT data, the harmonic powers are increased in the group of the HR cirrhotic !iver CT data and decreased in the group of the HR iron-overload !iver CT <lata. 
To test the described differences among the three groups of the HR CT data, elements of the ROC curves were calculated. Cut off points of 
Radio/ 011col 1999; 33(1): 9-14. 
Kur/Jel Sel al. / Detec/ion oj iron-overload mzd cirrhosis with CT 
Table 1. Statistical characteristics of the high-resolution liver CT <lata 
Mean minimal  CT Mean maximal  Mean density (HU)  Standard deviation  
value per  CT value per  of CT values (HU)  
sample (HU)  sample(HU)  


Controls (102 samples 
of HR liver CT <lata) Cirrhotic !iver patients (33 samples of 1-lR !iver CT <lata) 
Iron-overload patients 

(21 samples of HR -5.81 146.76 71.87 29.85 !iver CT <lata) 
Table 2. Results of the Fourier analysis. The power of the O. harrnonic equals the rnean density from the Table 1. Undistinguishing harrnonics in the range frorn 11. to 25. are ornitted as unirnportant 
I-larmonic 1. 2. 4. 5. 6. 7.8. 10. 

Cycles/rnm 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 
Wavelength(rnrn) 10 5 3.33 2.5 2 1.67 
1.25 1.11 
Controls (102 sarnples) 

Mean power (HU) 19.0 17.1 14.9 11.7 8.2 5.6 2.9 2.2 
St. deviation (HU) 11.2 10.1 8.5 6.8 5.0 2.3 1.8 
1.1 

Cirrhotic liver patients (33 sarnples) Mean power (HU) 25.9 22.9 18.0 14.5 10.0 6.3 4.4 3.3 2.6 2.0 

1.4 St. deviation (HU) 18.7 14.6 11.2 8.8 5.8 3.7 2.6 2.1 

Iron-overload patients (21 samples) 

Mean power (HU) 16.1 14.5 13.3 10.1 7.0 5.0 2.5 1.8 1.4 
St. deviation (I-IU) 9.1 8.1 8.1 5.4 4.0 2.9 2.1 1.5 1.1 0.9 


the highest P2 value and overall accuracy were selected as optimal. 
The results of the comparison between the HR cirrhotic !iver CT data and the control CT data are shown in Table 3. 
The cirrhotic !iver HR CT <lata can be distin­guished from the controls by the decreased mean density (<50 HU, P =37.28, p<0.0001) and by the increased power of harmonics from 0.1 to 0.9 cycles/mm (wavelengths from 10 to 1.1 mm)(p<0.01). Some harmonics showed an increased standard deviation of harmonic pow­ers among linear segments (p<0.01). 
The results of the comparison between the HR iron-overload !iver CT <lata and the control CT data are shown in Table 4. 
The iron-overload !iver CT <lata can be distin­guished from the controls by the increased mean density (>78 HU, P2=30.44, p<0.0001), decreased power of many harmonics from 0.1 to 


2.5 cycles/mm (wavelengths from 10 to 0.4 mm) (p<0.01). 
Discussion 

Spectral analyses proved to be a suitable tech­nique for discriminating the ultrasound images of normal livers and steatotic ones. The report­ed study included six cases (two normal and four with steatosis).5 The texture measure val­ues were compared with the corresponding biopsy scores and the results indicated the abili­ty of the spectral texture measure to discrimi­nate the two conditions and to estimate the severity of histological change. 
Radio/ Oncol 1999; 33(1): 9-14. 

Kurbel Set al. / Detec/ion oj iro11-overload and cirrlzosis with CT 
Table 3. ROC curve elements for the parameters distinguishing the cirrhotic !iver HR CT dala from the controls (P2>6.63, p<0.01) 
2 

Characteristics of HR CT data Sensitivity Specificity Accuracy x 
Cirrhotic Cul off point Controls liver (HU) 
Mean density  <  SO.O  ;,:  0.70  0.85  0.81  37.28  
Cycles/mm  Wavelength (mm)  Fourier analyses  
Mean  
power  0.1  10  ;,:  25.0  <  0.48  0.88  0.79  20.45  
(HU)  
0.2  5  ;,:  23.0  <  0.52  0.92  0.82  31.52  
0.4  2.5  ;,:  14.0  <  0.48  0.81  0.73  11.57  
0.5  2  ;,:  10.0  <  0.48  0.84  0.76  14.83  

0.7 1.43 
;,: 

< 0.55 0.74 0.69 8.84 
O.S 1.25 ;,: 3.5 < 0.45 0.86 0.76 14.88 
0.9 
1.11 
;,: 

< 0.55 0.7 2 0.67 7.49 
St. dev.  0.1  10  ;,:  12.5  <  0.45  0.87  0.7 7  16.23  
(I-IU)  
 
0.2  5  ;,:  10.0  <  0.55  0.7 2  0.67  7.49  
0.4  2.5  ;,:  7.5  <  0.52  0.81  0.74  13.79  
0.5  2  ;,:  5.2  <  0.52  0.78  0.7 2  10.88  

1.11 ;,: 1.6 < 0.45 0.86 0.76 14.88 
The same method was used to analyse tra­becular patterns in digitised wrist radiographic images. 6 Authors found in the group of 68 patients that three spectral indices permitted quantification of the cancellous bone structure, and detection of the age related structural bone changes. 
Another example is the reported study of the cross-sectional images of the posterior mandible.7 In these images, useful diagnostic information was in the range of 0.12 to 0.6 cycles/mm for the contact radiographs and the 3 mm cross-sections images. 

The idea of using high-resolution !iver CT values was based on previous reports that high­resolution CT abdominal imaging proved useful in the detection of the adjacent organs' invasion by the stomach cancer.8 
A CT slice thickness ranging from one to three millimetres is used in the chest CT imag­ing.9 The high-resolution CT of the lungs corre­lates well with the pathologic findings.10 It pro­vides detailed visualisation of the lung parenchyma.11 It is useful in differentiating sim­ilar patterns of abnormalities seen on chest radiographs, such as those seen in lymphangitic carcinomatosis and sarcoidosis, and in delineat­ing the extent of co-morbid lung diseases, such as emphysema and asbestosis. The characteris­tics of the margins of metastatic pulmonary nodules noted on histopathologic examination correlated well with their high-resolution CT findings (z04),12 while microscopic intravascu­lar tumour emboli and lymphangitic tumour spread were difficult to detect. 
Actual CT values or „pixel mapping" were found important in accurate diagnosing of small kidney angiomyolipomas.13 
The images in iron-overload patients differed from the controls by the increased mean densi­ty (>79 HU) and reduced power and standard deviation of power of many harmonics. It is in accordance with the reported observation that the mean !iver densities in patients with haemochromatosis are increased and in the range from 75 do 132 HU.14 The reduced pow-
Radio/ Oncol 1999; 33(1): 9-14. 

Kurbel Seta/./ Detec/ion of iron-overload a11d cirrlwsis with CT 
Table 4. ROC curve elements for the parameters distinguishing the HR iron-overload !iver CT data from the con­trols (X2>6.63, p<0.01) 
Characteristics of HR CT data Sensitivity Specificity Accuracy 2 
x 

Iron-Cut off point Controls overload (HU) 
Mean density :;, 79.0 < 0.38 0.98 0.87 30.44 
Cycles/mm Wavelength (mm) Fourier analyses 
Mean power 0.1 10 < 16.0 :;, 0.71 0.70 0.70 12.53 (HU) 
0.2 5 < 14.0 :;, 0.57 0.75 0.72 8.82 
0.4 2.5 < 10.0 ;,: 0.62 0.71 0.69 8.09 
1.43 < 3.5 ;,: 0.71 0.66 0.67 9.94 
0.7  1.43  <  4.4  ;,:  0.55  0.74  0.69  8.84  
O.S  1.25  <  2.4  ;,:  0.52  0.75  0.72  6.53  
0.9  1.11  <  1.9  ;,:  0.67  0.69  0.68  9.27  
1.0 1.1 2.5  1 0.91 0.4  <  1.3 1.2 0.3  ;,:  0.52 0.67 0.62  0.80 0.68 0.68  0.76 0.67 0.67  9.92 8.68 6.5  

St. dev. O.O 4 < 8.0 ;,: 0.57 0.78 0.75 11.02 (HU) 
0.4 
2.5 < 
;,: 
0.71 0.66 0.67 9.94 

O.S 2 < 3.7 ;,: 0.67 0.74 0.72 12.66 
0.75 1.43 < 
;,: 
0.67 0.68 0.67 8.68 

0.9 1.11 < 1.0 ;,: 0.57 0.76 0.73 9.5 


1  8.82  
0.91  8.09  
1.0  0.83  0.8  6.53  
0.77  7.00  
0.66  <  ;,:  -0.6  -0.7  -0.7  11.02  
0.62  9.30  
0.59  8.09  

0.55 0.4 6.50 
0.43 

0.42 6.53 
ers of harmonics suggest reduced variability in the densities of adjacent pixels. Possible inter­pretation is that an evenly diffused iron-over­load increases the mean density and simultane­ously blurs the normal variability of the high­resolution !iver CT data. 
The high-resolution CT data of the cirrhotic !iver in the present study differed from the con­trols by the low mean density (<50 HU), possi­bly because of the reduced partial volume effects, allowing visualisation of the infiltration of the fat in alcoholic !iver steatosis.15 
Acknowledgements 

The first author thanks Matija Bistrovic PhD, Stevan Doboš, Pero Strugacevac, Marko Jakic MD, Miroslav Volaric MD, Branko Dmitrovic MD, PhD, Nikola Micunovic MD, MSc, 
Radio/ 011co/ 1999; 33(1): 9-14. 

Kurbel S et a/./ Detection oj iron-overload and cirrlwsis with CT 
Dubravko Paulini MD and Aleksandar Vcev MD, PhD for their help. 
References 
l. Ritchins RT, Pullan BR, Lucas SB, Fawcitt RA, Best JJ, lsherwood 1, Morris AL An analysis of the spa­tial distribution of attenuation values in computed tomographic seans of !iver and spleen. J Comu/ Assist To111ogr 1979; 3: 36-9. 
2. 
Croxton FE, Cowden OJ, Klein S. Applied general statistics. Englewood Cliffs, New Jersy: Prentice­Hall Inc, 1967. p. 153-213. 

3. 
Grandis-Harrison D. Ql abacus. In: Berry S, edi­tor. Ql user guide. Cambridge, Great Britain: Sin­clair Research; 1984. p. 1-24. 

4. 
Goldman L. Quantitative aspects of clinical rea­soning. In: Braunwal E, Isselbacher KJ, Petersdorf RG, Wilson JO, Martin JB, Fauci AS, editors. Harri­


11th 
son's principles oj interna/ medicine. ed. New York: McGraw-Hill; 1987. p. 5-11. 
5. 
Khoo BCC, McQueen MPC, Sandle WJ. Use of texture analysis to discriminate between normal livers and livers with steatosis. J Biomed Eng 1991; 13: 489-94. 

6. 
Wigderowitz CA, Abel EW, Rowley DI. Evaluation of cancellous structure in the <listal radius using spectral analysis. Ciin Ortlwp 1997; 335: 152-61. 

7. 
Chen SK, Hollender L. Frequency domain analy­sis of cross-sectional images of the posterior mandible. Oral Surg 0ml Med Oral Pathol 1994; 77: 290-5. 



8. 
Tsuburaya A, Noguchi Y, Matsumoto A, Kobayashi S, Masukawa K, Hori-Guchi K. A pre­operative assessment of adjacent organ invasion by stomach carcinoma with high-resolution com­puted tomography. Surg Today 1994; 24: 299-304. 

9. 
Napel S, Rubin GO, Jeffrey RB Jr. STS-MIP: a new reconstruction technique for CT of the chest. J Comp Assist Tomogmphy 1993; 17: 832-8. 

10. 
Swensen SJ, Aughenbaugh GL, Douglas WW, Myers JL. High-resolution CT of the lungs: find­ings in various pulmonary diseases. Am J Roentgenol 1992; 158: 971-9. 

11. 
Spillane RM, Shepard JA, DeLuca SA. High-reso­lution CT of the lungs. Am Fa111 Physician 1993; 48: 493-8. 

12. 
Hirakata K, Nakata H, Haratake J. Appearance of pulmonary metastases on high-resolution CT seans: comparison with histopathologic findings from autopsy specimens. AJR Am J Roentgenol 1993; 161: 37-43. 

13. 
Takahashi K, Honda M, Okubo RS, Hyodo H, Takakusaki H, Yokoyama H, et al. CT pixel map­ping in the diagnosis of small angiomyolipomas of the kidneys. J Comp Assist Tomography 1993; 17: 98-101. 

14. 
Goldberg HI. Recognition of hepatocellular disor­ders by computed tomography. In: Moss AA, Goldberg HI, Norman D, editors. Interventional radiologic techniques: computed tomography and ulra­sonography. New York: Academic Press; 1981. p. 212-8. 

15. 
Crawford JM. The liver and the biliary tract. In: Cotran RS, Kumar V, Robbi11s SL, editors. Robbins pathologic basis oj disease. 511' ed. Philadelphia: WB Saunders; 1994. p. 831-96. 


Radio/ Onco/ 1999; 33(1): 9-14. 

Radio/ Oncol 1999; 33(1): 15-21. 
Standardized image documentation in nuclear medicine 
Karl H. Bohuslavizki, Ralph Buchert, Janos Mester, Malte Clausen 
Department oj Nuclear Medicine, University Hospital Eppendorf Gennany 
There are no generally accepted standards jor image docu111entation in every day nuclear medicine practice. This is a problem whenever hardcopies jrom other centers are to be re-intcrpretcd or comparcd to prescnt images oj thc same patient. In ordcr to support image rcading by a third party proposals oj documentation neccssary within images are elaborated. Exmnples oj image documentation oj the most jrcquently per­jormed nuclear medicine studies are given, i.e. /ung scintigraphy, thyroid scintigraphy, bone scintigraphy both in planar and in SPECT-technique, renal junction scintigraphy, myocardial perjusion scintigraphy, and positron ernission tmnography. These exmnples are intended to stimulate discussion within the nuclear medicine community about the content oj documentation necessary in nuclear medicine irnages. 


Key words: nuclear medicine; diagnostic imaging; medica! records-standards; minimal requirenzents 
Problems of image documentation in nuclear medicine 

There are no generally accepted standards for image documentation in every day nuclear medicine practice. This is a problem, when­ever hardcopies from other departments/clin­ics/institutions have to be re-interpreted or compared to present images of the same patient. This problem is independent of the image quality itself. Even best image guality may be insufficient for interpretation, if essential information is missed. In conse­guence, incomplete image documentation may render interpretation difficult or even 
Received 31 July 1998 



Accepted 15 October 1998 Correspondence to: Dr. Karl H. Bohuslavizki, Depart­ment of Nuclear Medicine, Univer-sity Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Ger­many. Phone: +49 40 4717 4047; Fax: +49 40 4717 6775; E-mail: bohu({omedsph2.uke.uni-hamburg.de 
impossible. Thus, the main task of the work­ing group „Standardized Image Documenta­tion" of the German Society of Nuclear Medi­cine is to define guidelines for minimal reguirements of nuclear medicine image doc­umentation. This group has gained experi­ence in this field for the last three years. Their proposals have been discussed on sev­era! national conferences and published in 
4 

different journals.1-The members of the working group are co-operating with corre­sponding working groups of various national nuclear medicine societies. 
The examples presented below should be extensively discussed in order to maintain a consensus about them. These generally accepted standards could than support the manufacturers of nuclear medicine compu­ters to implement the accepted requirements within their software. However, the standard­ization of image documentation should be 
Bohuslavizki KH et a/. / Standardized image docwnentation i11 nuc/em· medicine 
considered as definitely independent of the ongoing standardization of nuclear medicine procedures. Furthermore, the elaborated pro­posals should not limit the definition of indi­vidual layout or individual extensions in image documentation. 
Examples of images 
The presented examples of typical nuclear medicine studies are partly representing the results of consensus conferences held as pre­congress meetings prior to the last three national congresses of nuclear medicine in Germany. The subcommittee considers them in a presented form as an initial point for an intended discussion within the European Nuclear Medicine Community. They are designed to cover the requirements of every day routine studies. The limitation of labeling to the minimum necessary for understanding of the image content by third parties is one of the declared intentions of the subcommittee. However, individual extensions of these min­imal requirements by particular institutions should be permitted definitely. 
Severa! data should be documented clearly on all nuclear medicine studies, Le. patient name, date of birth, date of the study, and institution in which the study was per­formed. The injected amount of radioactivity and the tracer used are probably the most important study related data. The interpreta­tion of the image should be supported by the display of the color bar used. Parameters derived from regions-of-interest should be supported by documentation of the underly­ing ROis. The image sequence with the time of individual images should be documented for image sequences. 
For most often performed types of studies the following specific requirements should be fulfilled in addition to the above mentioned basic requirements. 
Lung 
Thorough labeling of the views is mandatory in Jung scintigraphy. Ventilation and perfu­sion studies should be clearly distinguished by appropriate labels (Figures 1 and 2). Docu­mentation may be completed by denoting the side-related perfusion as well as the position of the patient both at the time of injection and during acquisition. 
Thyroid 
Labeling of the jugulum and of the Tech­netium-99m-uptake value in percent is essen­tial in documentation of thyroid scintigraphy. It is recommended to complete the documen­tation by the matrix size, the display of the reference scale, and the time interval between injection and acquisition. It may be helpful to identify findings of palpation with­in the image (Figure 3). 
Bone 
Whole body bone seans should be document­ed at least in two directions both at two intensity levels, the first optimized for the rib s and the second for the spine (Figure 4). 
Myocardiwn 
For myocardial perfusion SPECT, the whole left ventricular myocardium has to be docu­mented. Short axis, horizontal and vertical long axis slices should be presented both as stress and as rest images. An optimized arrangement of identical slices of the stress and resting study within the same image is recommended. Moreover, information on the position of the patient during acquisition and anatomical labeling of the slices are required. Pictograms or data about the type of the stress may support interpretation of the images as well (Figure 5). 
Radio/ Oncol 1999; 33(1): 15-21. 

Boliuslnvizki KH et a/. / Stnndnrdized imnge documentntion in nuclenr medicine 

Clinics / Department / Institute 
Musterman , Otto, date of birth: 06-16-64, date of study: 05-29-98 
100 



Ventilation: 50 MBq Tc-99m-MDP 

L D R Aerosol 

RPO LPO 
Figure l. Ventilation scintigraphy of the lung. 
Clinics / Department / Institute 
Musterman , Otto, date of birth: 06-16-64, date of study: 05-29-98 

100 

Perfusion: 
100 MBq 
Tc-99m-MAA 

L D R R v L 

-o 

RPO LPO 55 % 45 % 
Figure 2. Perfusion scintigraphy of the lung. 
Rndiol Onco/ 1999; 33(1): 15-21. 

Bohuslavizki KH et al. / Standardized image documentation in nuclear medicine 
Clinics / Department / Institute 
Musterman , Otto, date of birth: 06-16-64, date of study: 05-29-98 

100 
100 MBq 
10 cm Tc-99m-Pertechnetate 128 x 128 Matrix 
rT 
R v L 
TcTU = 6.2 % (20 min p.i.) 
-o 
* Palpation 
R v L 
Figure 3. Quantitative thyroid scintigraphy. 

Clinics / Department / Institute 
Musterman, Otto, date of birth: 06-16-64, date of study: 05-29-98 



-o 

R V L R V L L D R L D R 
Figure 4. Whole body bone scan. 
Radio/ Oncol 1999; 33(1): 15-21. 

Bohuslavizki KH et al. / Standardized image documentntion in nuclenr medicine 
Clinics / Department / Institute 
Musterman , Otto, date of birth: 06-16-64, date of study: 05-29-98 
Supine Stress: 200 MBq Tc-99m-XY Rest: 600 MBq Tc-99m-XY 100 

Stress ant. 
sep. ([)lat. Rest 
post. 
Stress ant. 
.api 
__.,/ Resi 
post. 
. 
ap1. Stress 
sep ( .lat. 


.t 
\ Resi 

-o 
Figure 5. Myocardial perfusion scintigraphy. 
Clinics / Department / Institute 
Musterman , Otto, date of birth: 06-16-64, date of study: 05-29-98 
700 .­
--------.==..::::, 


100 ._:.J?";l,' 

.-600 
..;,, ...,} ; , .. :, , . 
Left 500 -o-Right 

L D R 
00««-20«« 
c:: 400 

3«-5« 
20««-40«« 40««-60«« 
t: 300 
200 
,oo 

22«-24« 
7«-10« 10«-14« 14«-18« 18«-22« 
Supine 
II .2.. J1. 20 22 28 
o •• .-_ 
L: 

51 % time[min] 
49% 

100 MBq Tc-99m-Pertechnetate before after Clearance according to Oberhausen: 1341 ml/min/1.73 sqm 1 
-o 

voiding voiding Lower limit: 264 ml/min/1.73 sqm 
Figure 6. Rena! function scintigraphy. 
Radio/ Oncol 1999; 33(1): 15-21. 

Bohuslavizki KH et al. / Standardized image docum.entation in nuclear rnedicine 
Clinics / Department / Institute 
Musterman , Otto, date of birth: 06-16-64, date of study: 05-29-98 600 MBq Te -99m-HDP 

v 
R L 
D 
cranial 

coronal 
cranial 
R L 
caudal 

Clinics / Department / Institute 
Musterman , Otto, date of birth: 06-16-64, date of study: 05-29-98 
400 MBq F-18-FDG 100 coronal 




cranial 
anterior 
posterior R 
caudal 




-o .. 
Figure 8. Coronal slices of positron emission tomography. 
Radio/ Oncol 1999; 33(1): 15-21. 

Bohuslnvizki KH et ni./ Stnndnrdized imnge documentation in nuc/enr medicine 
Clinics / Department / Institute 
Musterman , Otto, date of birth: 06-16-64, date of study: 05-29-98 400 MBq F-18-FDG 
100 


V LL D RAO 

-o 
Figure 9. M!Ps of positron emission tomography. 
Function oj the kidneys 
Split renal function is the main data that should be documented within the image. Apart from numeric data on clearance, the method and age-dependent lower limits should be given in renal scintigraphy. After diuretics the documentation of the tirne of application, the curve and the effect of the diuretics in percent are essential (Figure 6). For the Captopril scintigraphy a separate doc­umentation is recommended. 
SPECT /PET 

Regarding tomographic studies both the direction of the slice sequence and an anatomical labeling of the slices should be presented within the images (Figures 7 and 8). The interpretation of images may be sup­ported by the use of pictograms. PET studies may be displayed as maximum intensity pro­jections (MIPs) for overview (Figure 9). 
R V L LAO 
Conclusion 

Standardized image documentation in nucle­ar medicine is mandatory in order to ease image reading by a third party. Suggestions given above should be extensively discussed among nuclear medicine physicians in order to maintain a consensus about them. 
References 

l. Bohuslavizki KH, Mester J, Clausen M: Bild­
beispiele zur standardisierten nuklearmedizinis­chen bilddokumentationen. Nuklenrmedizin 1997; 36: 53-4. 

2. 
Clausen M, Bohuslavizki KH, Mester J, Henze E: 

Vergleichbearkeit nuklearmedizinischer bilddoku­mente. Der Nuklearmediziner 1996; 19: 213-4. 


3. 
Mester J, Bohuslavizki KH, Clausen M, Henze E: 

Empfehlungen zur standardisierung nuklearmedi­zinischer bilddokumentationen. Der Nuklennnedi­ziner 1997; 20: 197-9. 


4. 
Bohuslavizki KH, Buchert R, Mester J, Clausen M: 


Nuklearmedizinische bilddokumentation 
arbeitsgemeinschaft standardisienmg der DGN. Der Nuklenr111edizi11er 1998; 21: 222-4. 
Radio/ Oncol 1999; 33(1): 15-21. 


Radio/ Oncol 1999; 33(1): 23-6. 
Detection of lymph node metastasis from osteosarcoma with 99mTc-MDP scintigraphy. Case report 
Daniela Ilic1 , Georgi Zafirovski2, Nina Simova1 , Nikola Zisovski3 , Sofijanka Glamocanin3,Vesna Janevska4, Cvetka Tolevska5 , Olivija Vaskova1, Milan Samardziski2 
1Institute oj Pathophysiology and Nuclear Medicine, 2Clinic jor Orthopedic Surgery, 3 Clinic jor Pediatrics, 4Institute oj Pathology, 5Institute oj Radiotherapy and Oncology, Medica/ Faculty Skopje, Macedonia 
Osteosarcoma usually spreads via the blood stream, developing pulmonary and skeleta/ metastasis. Rarely, the disease spreads via the lymphatics. We are reporting a case when radionuclide bone imaging detects lymph node metastasis jrom osteosarcoma. 
Key words: bone neoplasms -radionuclide imaging; osteosarcoma; lymphatic metastasis; technetium Te 99 m medronate 
Introduction 

Osteosarcoma, as other malignant bone tumors demonstrates active bone turnover and therefore reveals intense uptake on bone scintigraphy.1 Bone-seeking radiopharmaceu­ticals occasionally demonstrate intense uptake in soft tissue metastasis from osteosarcoma (lung, brain, renal, lymph 
4 

nodes).2-This tumor spreads usually via the blood stream with development of pul­monary and skeleta! metastasis. 
Received 2 July 1998 Accepted 15 December 1998 
Correspondence to: Daniela Ilic, MD, MSc, Institute of Pathophysiology and Nuclear Medicine, Vodnjans­ka 17, 91000 Skopje, Republic of Macedonia; Phone/ Fax: +389 91 112831. 
Case report 

The patient, male at the age of 14, first pre­sented two years before this report. He had been complaining of painful, livid swelling in his left calf for 4 months until he was referred to us. He was seen by pediatric oncologist after radiography, bone scintigra­phy, fine needle aspiration and open biopsy had been perfonned. Histology finding con­firmed osteosarcoma of the left tibia. 
Plain radiography of the left leg demon­strated a poorly-defined lesion located cen­trally in the left proximal metaphysis of the tibia, extending into the shaft. The lesion revealed a permeable pattern of destruction with proliferation, extensive sunburst spicu­lation, mineralization of the tumor, lumps, clouds and segments of ossified matrix. The 
Ilic Det a/. j Lyrnph node matastasis and 99mTc-MDP scintigraphy 

Figure l. Pain radiography of the left tibia (in the period of the initial diagnosis). 
radiographic finding with these features strongly suggested high grade osteosarcoma (Figure 1). 
Bone scintigram with 99mTc-MDP revealed inhomogenously increased uptake of bone radiotracer in the upper third of the left tibia with extension into the soft tissue adjacent to the bone (Figure 2). 
After the confirmation of osteosarcoma, chemotherapy with T6 protocol for solid tumors was commenced because the parents refused surgery. T6 induction and mainte­nance chemotherapy for solid tumors was used for 14 months after the initial diagnosis. Before the completion of chemotherapy (exactly one year after histologic confirma­tion of osteosarcoma) above-knee amputation was done. Three months after the surgery, enlarged lymph node in the left groin was noticed. CT scan of the left groin demonstrat-
Radiol Oncol 1999; 33(1): 23-6. 

, 



• 
Figure 2. 99mTc-MDP bone scan of the primary tumor. 


Figure 4. 99mTc-MDP bone scan after chemotherapy and surgery. 

1/ic Det ni./ Lymph node mntastasis nnd 99mTc-MDP scintigrapliy 

Figure 3. CI of the pelvis (region of the left groin). 
ed the mass (lymph node) with punctate cal­cification (Figure 3). 99mTc-MDP bone scan did not show any focal hot spots throughout the skeleton, except an area of increased radiopharmaceutical activity loca ted closely to the trochanter minor of the left femur, cor­responding to the enlarged lymph node {Fig­ure 4). The lymph node was removed and microscopic section {hematoxylin and eosin, x 400) revealed a metastatic deposition of osteosarcomatous tissue. There was a mes­enchimal, osteoblastic stroma in which a deposition of osteoid and calcified osseous trabecullae (arrow) were found {Figure 5). 
After the removal of the lymph node exter­nal beam radiotherapy of 50 Gy in the region of the left groin was applied. Recently after the termination of the radiotherapy, chest X­ray revealed pulmonary metastases. The child was in very bad condition and as he did not respond to radiotherapy at ali, orthopedic surgeon, radiotherapist and pediatrician oncologist decided not to apply any other treatment. One month later (20 months after the onset of the disease) the patient died. 
Discussion 

The value of bone radionuclide imaging in the management of osteosarcoma is well established. Bone scan is quite useful in the delineation of the extent of the primary lesion, as well as in the follow up studies.5­Local recurrence or skeletal metastasis are detected before they are radiographically apparent. 
Osteosarcomatous lesions that spread via the lymphatics are very uncommon. Litera­ture review revealed 2.7-11.4% of lymphatic 
Rndiol Oncol 1999; 33(1): 23-6. 

Ilic Det al. / Lymph node matastasis and 99mTc-MDP scintigraphy 

Figure 5. Microscopic section of the removed lymph node. 
7 
metastases in patients with osteosarcoma.lts presence is a bad prognostic sign of the disease as none of these patients survived 5 
8 years.
Lymph node metastasis accumulate bone­seeking radiopharmaceutical avidly so 99mTc-MDP bone scan could be used in the assessment of any spreading (hematogenous or lymphatic) of osteosarcoma. 
References 
l. Donohoe KJ. Selected topics in orthopedic nuclear medicine. Orthopedic Clinics oj Norih America 1998; 29: 85-101. 
2. Gilbert AL, Weiss MA, Gelfand MJ, Hawkins HH, Nishijama H, Aron BS. Detection of renal metas­tasis of osteosarcoma by bone scan. Ciin Nucl Med 1983; 8: 325-6. 
Radio/ Oncol 1999; 33(1): 23-6. 
3. 
Heyman S. The lymphatic spread of osteosarcoma shown by Tc-99m-MDP scintigraphy. Ciin Nucl Med 1980; 12: 543-5. 

4. 
Jefree GN, Price CHG, Sissons HA. The metastat­ic pattern of osteosarcoma. Br J Cancer 1975; 32: 87-107. 


5. Mandell GA, Harcke HT. Pediatric bone scanning. In: Collier BO, Fogelman I, Rosental L, editors. Skeleta/ nuclear medicine. St Louis: Mosby; 1996. p. 339-41. 
6. Me Killop JH: The bone scan in primary bone tumors and marrow disorders. In: Fogelrnan I, edi­tor. Bone scanning in clinical practice. Berlin: Springer Verlag; 1987. p. 61-72. 
7. 
Caceres E, Zaharia M, Antaleen E. Lyrnph node rnetastases in osteogenic sarcorna. surgery. 1969; 65: 421-2. 

8. 
Andreev I, Raicev R: Kostoobrazuvasti turnori. In Andreev I, editor. Tumori na kostite. Sofija: Izdatel­ska kornpanija K&M, Medicina i fisklutura; 1993. p. 36-61. 



Radio/ Onco/ 1999; 33(1): 27-33. 
A correlation of NK cytolitic test and BLT esterase test in determining activity of NK cells, stimulated by tumor target cells 
Alojz Ihan 
Institute oj Microbiology and Immunology, Medica/ Faculty, University oj Ljubljana, Slovenia 


We examined the granule exocytosis in natura/ kil/er (NK) cel/s by measuring N-benzyloxycarboxy-L-lysine esterase activity. As a source oj NK cel/s we used bujjy-coat isolated NK ce/ls or peripheral blood lympho­cytes (PBL). The exocytosis was triggered by incubating cel/s with ionomycin/PMA or by NK cell suscepti­ble tumour target cells K562. When we stimulated purijied NK ce/ls with tumour target cel/s, a close corre­lation (Corr=0.84) oj cytolitic NK test results and BLT test results was obtained. We may cone/ude that BLT test can provide a valuable tool to discriminate jurther NK celi dejiciency in patients with low cytolitic NK test results. 
Key words: killer cells, natura/; exocytosis, celi degranulation 
Introduction 

Secretory granules of lymphocytes may be important organelles, serving to concentrate and store different enzymes, cytolitic pro­teins and biologically active molecules. It is possible that exocytosis of granules plays an important role in cytolitic activity of cytotox­ic T lymphocytes (CTL). Trypsin-like serine esterase (BLT esterase) was described as an easily detectable biochemical marker of gran­ules.1 It was shown that secretion of this 
Received 11 February 1998 
Accepted 18 June 1998 Correspondence to: Assist. Prof. Alojz Ihan MD, PhD, Institute of Microbiology and Immunology, Medica! Faculty, University of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia. Tel: +386 61 140 30 42, Fax: +386 61 302 895. 
enzyme is triggered by incubating CTL with antigen-bearing target cells or with immo­bilised anti-TCR monoclonal antibodies -in both cases the crosslinking of T-cell receptors (TCR) is required. The exocytotic process can be also triggered by activation of protein kinase C (with phorbol esters) and by simul­taneous translocation of calcium through CTL plasma membrane with Ca2+ ionophores.2 It has been shown that external Ca2+ is obligatory for both the TCR-triggered and for the phorbol ester/ Ca2+ ionophore ­triggered exocytosis, because Ca2+ chelators (EDTA, EGTA) and Ca2+ channel blockers (nifedipine, verapamil) inhibit exocytosis.3 
Natura! killer cells (NK cells) are a sub­population of lymphocytes, whose function has been defined to date by their ability to mediate the in vitro destruction of certain 

Ihan A / Natura/ ki/ler cel/s 
neoplastic and virally infected cells in a rapid, non-MHC restricted fashion. Because NK cells do not express antigen receptors they must recognise their targets in a way, that differs from T cells antigen recogni­tion.4,5 Nevertheless, the killing mechanisms of both cell types may be similar and they include granule exocytosis that releases enzymes, cytolitic proteins and biologically active molecules into areas bounded by the close apposition of effector and target cell plasma membranes.6, 7 
In our experiments, we provided an evi­dence that the BLT esterase, besides the use for monitoring CTL exocytosis, can also be used as a biochemical marker of NK cell granule exocytosis. We used BLT-esterase assay for monitoring NK cell exocytosis. As a source of NK cells we used buffy-coat isolat­ed NK cells or buffy-coat isolated lympho­cytes (PBL) as well. The exocytosis was trig­gered by incubating cells with ionomycin/ PMA or by NK cell susceptible tumour target cells K562. 
Materials and methods 

Separation oj human peripheral blood lympho­cytes (PBL) 
Buffy coats from normal blood donors were obtained with the kind co-operation of Trans­fusion Centre in Ljubljana. Buffy coats were diluted with two parts of sterile PBS and overlayered on Ficoll-Isopaque. Gradients were spun at room temperature for 20 min at 250 g. Cells (PBL) were subsequently harvest­ed from above with a Pasteur pipette and washed three times. An aliquot from each sample was then suspended in trypan-blue solution for counting in a counting chamber. 
NK cell isolation 
Ficoll-isolated PBL cells were first incubated in nylon-wool columns in order to remove 
Radio/ Oncol 1999; 33(1): 27-33. 

adherent cells (monocytes, B lymphocytes). Columns consisted of 10-ml plastic syringes, filled with 0.5 g of nylon wool. Each column was pre-washed with 30 ml of warm RPMI (37 ° C) and loaded with 1 ml of PBL suspen­sion. Maximal cell load/column was 100 mil­lion cells. After 1 hr incubation at 37 ° C in a 5% CO2 humidified atmosphere, non-adher­ent cells were gently washed out with 20 ml of wann RPMI (37 ° C). For further NK cell iso­lation, a commercially obtained preparation of polyvinylpyrrolidone-coated silica (Percoll, Pharmacia, Uppsala, Sweden) was first made isotonic for use with living lymphocytes. 1.5 M phosphate-buffered saline (PBS) was added to the stock solution of Percoll in a ratio of one part PBS to nine parts Percoll. For subsequent use, this stock (100%) was diluted with 0.15 M PBS or cell culture medi­um for cell separations. Discontinuous gradi­ents were generated in 12 ml sterile plastic centrifuge tubes, which had been previously wetted with a small amount of serum to allow even flow of the Percoll. Non-adherent PBL cells, obtained from nylon-wool columns, were resuspended in 2 ml of 100% Percoll and successively less dense solutions layered carefully on top in 2 ml aliquots. Gra­dients for lymphocytes were run between 60% and 40%, with a 10% differential between layers. Once generated, gradients were spun for 10 min at 450 g. After centrifugation, dis­tinct bands could be observed at the various interfaces. Gradients were harvested from above with a Pasteur pipette, and the cellular composition of each fraction was evaluated flow cytometrically. Cell preparations, con­taining more than 85% of NK cells (CD3­CD56+ cells) were considered NK cell homogenous and were used in subsequent experiments. 
NK-enriched PBL cells 
Ten ml of heparinised blood was diluted 1/2 with RPMI and layered on Ficoll-Isopaque. 

lhnn A / Nntural ldller cel/s 
Isolated PBL cells were further separated by Percoll centrifugation as described above. Low density cells were collected with a Pas­teur pipette and used for NK test and BLT-E release assay. 
Short-term ce/l culture with IL-2 
Isolated cells were (2x106 cells/rnl) cultured in a 24-well plate in the presence of 100 units/ml of recombinant human IL -2 (Sigma). After 18 hours of incubation (37 ° C in humidi­fied 5% CO2 atmosphere) the cells were recovered and used for subsequent studies. 
NK test 
Effector cells were tested against 51 Cr­labelled K562 target cells. K562 cells (5x106) in 0.5 ml mediurn were labelled by the addi­tion of 100 (Ci sodiurn chromate (Na/1CrO4, sp. act. 150-500 (Ci/mg) and incubated at 37 ° C for 1 hr. The cells were then washed three tirnes in rnediurn and resuspended to a concentration of 1 x 106 cells/ml in complete medium. Effector cells were prepared at a concentration of 5 x 106/ml and added to each well of the microtiter plate in a volume of 100 (l. Triplicate cultures were established in U-bottomed rnicrotiter plates in a total vol­ume of 100 (1, diluted in complete mediurn to obtain target/effector ratio (T/E) ranging from 1/4 to 1/64. Hundred µl of target cells were then added to effector cells. Plates were incubated for 4 h at 37 ° C in a 5% CO2 hurnid­ified atmosphere, and released radioactivity was measured in a gamma counter. The per­centage cytotoxicity was calculated by the equation: % cytotoxicity = (cpm experimental release -cpm spontaneous release) x 100 / (total radioactivity incorporated into cells). Spontaneous release was determined by incubating the target cells in medium alone. To compare the results at different E/T ratios, data are expressed in lytic units (LU). One LU represents the number of effector cells required for lysis of 20% of target cells. LU were determined from log-linear plots of the data points, and are reported at LU20/107 effector cells.8 
BLT test 


The amount of secreted BLT esterase was measured using 1 x 105 cells (PBL or NK cells) in 0.1 ml of celi culture medium in the 96-well microtiter plate in the presence of tumour target cells (K-562) or pharmacologi­cal stimuli -PMA (10 ng/ml) / A23187 (0.5 ug/ml). After 4-hr incubation at 37 ° C in a 5% CO2 humidified atmosphere, cells were resuspended by gentle pipetting and cen­trifuged at 200 g for 5 min. Fifty µl aliquots of supernatant were used to assay BLT esterase activity. Total cel!ular content of the enzyme was determined using 0.1% Triton X-100 solu­bilized cells. Determinations were carried out in triplicate. Data are presented as specific percentage of enzymatic activity released which was calculated from the equation: % release = 100 x (E-S) / (T-S), where E repre­sents the number of enzyme units in the supernatant of the experiment well, S the number of enzyme units in the supernatants of the well containing no stimuli, and T the total number of units of BLT esterase per well. The total BLT esterase content in target cells K-562 was always less than 5% of that in PBL or NK cells. For measuring BLT esterase activity, 0.05 ml of culture medium was mixed with 0.95 ml of BLT solution (0.2 mM BLT, 0.22 mM DTNB in PBS, pH 7.2). The mixture was incubated at 37 ° C for 20 min and the reaction was stopped by adding 5µ1 of 0.1 M PMSF, which was dissolved in dimethylsulfoxide. The solution was diluted by 1 ml of PBS and absorbency at 412 nm was measured in comparison to a blank solu­tion (cel! culture medium) that was treated exactly as experimental. 
Rndiol 011co/ 1999; 33(1): 27-33. 

Ihan A j Natura/ kil/er cel/s 
Flow cytometry 
Flow cytometric analysis was performed by a fluorescence-activated celi sorter (FACSort, Becton Dickinson, Mountain View, CA, USA). A two-parameter analysis was per­formed to determine the proportion of CD3­CD56+ cells in samples.9 Monoclonal anti­bodies (CD3-FITC, CD56-PE) were purchased from Becton Dickinson (Mountain View, CA, USA). 
Statistical analysis 
Correlation analysis was calculated by using a standard analysis package Quattro pro (Borland International, USA). 
Results 
For determining the tirne course of BLT-E secretion by NK cells, a secretion of Percoll­isolated NK cells was triggered by synergistic action of PMA and ionophore A23187 or NK cell susceptible tumour cells K562. Treatment of NK cells with PMA and A23187 results in faster release of granule enzyme than during incubation with K562 cells (Figure 1). When NK cells were incubated with K562 cells, BLT-E measurement reached stable values in 3-4 hours. Therefore a 4-hr incubation tirne was chosen for performing further experi­ments. 
As a granule enzyme release is thought to be involved in NK cell cytotoxicity, we com­pared the influence of K562-induced NK cell triggering on BLT-E secretion and NK-cyto­toxicity in healthy blood donors. In Figure 2 we present the data of experiments done on peripheral blood mononuclear cells. A poor correlation (Corr=0.54, SE =3.2) results most probably from low BLT secretion levels that are very close to a background release levels. Because NK cells represent only a minor part in PBL, BLT-E secretion results can be improved by using purified NK cells. Our 

Radio/ Oncol 1999; 33(1): 27-33. 

80 70 
,R60 'a;' 50 
)i'l 40 . 30 . 20 10 
o 
o 0.5 1 2 3 4 5 6 
incubation tirne (hr) 
Figure l. The tirne course of BLT esterase secretion by purified NK cells stimulated with !'MA/ A23187 (empty columns) or tumour target cells K562 (black colunms). 1 x 105 purified NK cells were incubated in the presence of !'MA/ A2387 or tumour target celi s K562. After incu­bation periods indicated on the abscissa, BLT esterase activity in the supernatant was measured. BLT esterase activity is presented as a percentage of maximal BLT release. Each point reprcsent an average of at least four experiments, made in triplicates. 
50 60 80 90 100 "110 120 "130 
Figure 2. A correlation of NK cytolitic test and BLT-E test results in pcripheral blood lymphocytes (PBL), stimulat­ed with tumour targets. Peripheral blood lymphocytes of normal blood donors were stimulated with tumour target cells K562 at different effcctor/target ratios (E/T = 1/4 ­1/64 ). After 4 hour stimulation, supernatants were col­lected for cytolitic NK celi test and BLT-E release test as well. NK tests results are expressed in lytic units (LU) and BLT-E test results are expresscd as percentages of maximal BLT-E rclease from effector cells. Each point represent an averagc of at least four experiments, made in triplicates. 
results (Figure 3) demonstrate that detectable BLT-E secretion results can be achieved in experiments done on pure NK cells. Also a correlation (Corr 

0.84, SE=l.28) of NK cell cytotoxicity and BLT-E secretion results was improved substantially. 


I/11111 A /Natura/kil/er cel/s 

120 140 '160 180 200 220 240 . 260 

Figure 3. A correlation of NK cytolitic test and BLT-E test results in isolated NK cells, stimulated with tumour tar­gets. Purified NK cells of normal blood donors were stimulated with tumour target cells K562 at different effecto1/target ratios (E/T = 1/4 -1/64 ). After 4 hour stimulation, supernatants were collected for cytolitic NK celi test and BLT-E release test as well. NK test results are expressed in lytic units (LU) and BLT-E test results are expressed as percentages of maximal BLT-E release from effector cells. Each point represents an average of at least four experiments, made in triplicates. 

120 '140 160 180 200 220 240 260 

Figure 4. A correlation of NK cytolitic test and BLT-E test results in NK-enriched peripheral blood lymphocytes, stimulated with tumour targets. NK-enriched of normal blood donors were stimulated with tumour target cells K562 at different effector/target ratios (E/T = 1/4 -1/64 ). After 4 hour stimulation supernatants were collected for cytolitic NK cell test and BLT-E relcase test as well. NK tests results are expressed in lytic units (LU) and BLT-E test results are expressed as percentages of maximal BLT-E release from effector cells. Each point represents 
an average of at least four experiments, made in tripli­cates. 
Flow cytometrical evaluation of NK­enriched PBL revealed that 3-4 x enrichment of NK cell concentration was usually obtained, with NK celi content ranging from 40 to 65%. As shown in Figure 4, maximal NK lysis reached 250 LU while maximal BLT­E release was 10% of total BLT-E content of peripheral blood Iymphocytes. The correla­tion of NK cell cytotoxicity and BLT-E secre­tion results(Corr = 0.79, SE=0.97) was close to correlation in experiments performed on purified NK cells. 
Discussion 

A vast majority of studies on granule exocy­tosis has been performed on cytotoxic T clones, while NK cell clones have been stud­ied less frequently.10-13 For studying exocyto­sis we used purified NK cells instead of cloned cells. An advantage of purified cells is that they better represent an in vivo physio­logical situation while cloned cells may have altered many of their phenotypic characteris­tics during long term in vitro cultivation. A disadvantage of studies on purified NK cells may be, besides laborious isolation proce­dure, a weak exocytotic activity of purified NK cells. Because previous reports 11 indicat­ed that IL-2 can enhance, in a cytokine con­centration-dependant manner, NK cell-medi­ated cytotoxicity, we established a short-term (18 hours) culture of purified cells to obtain exocytotic activities well distinct from back­ground values. 
Using the assay system described, it is possible to study enzyme granule exocytosis of NK cells. Because BLT esterase was described by many authors as an easily detectable biochemical marker of cytolitic granules, we used BLT-E release assay to detect NK celi exocytosis. It was shown that BLT-E is released by immune cells, capable of cell-mediated killing -including CTL and NK cells as well. In vivo, CTL release cytotoxic granules after they recognise surface anti­gens presented by MHC I molecules on the surface of target cells (virus infected or tumour cells). In vitro a similar process can be triggered by incubating CTL with antigen­bearing target cells or with immobilised anti­TCR (CD3) monoclonal antibodies -in both 
Radio/ 011col 1999; 33(1): 27-33. 

Ihan A / Natura/ kil/er cel/s 
cases the crosslinking of T-cell receptors (TCR) is required. On the other hand, NK cell utilise, in vivo two distinct mechanisms to trigger exocytosis. One, termed antibody­dependent cellular cytotoxicity (ADCC), utilises NK celi surface Fc-receptors and anti­bodies directed against the target-cell anti­gens. The other involves direct interaction between NK cells and their target cells and utilises NK cell receptors that have not yet been fully characterised.14, 15 In vitro, a process of NK killing can be demonstrated by incubating NK cells with antibody coated (ADCC killing) or some tumour target cells (K562 cells). 
The exocytotic process in both celi types (CTL and NK) can be also triggered in vitro by the activation of protein kinase C (with phor­bol esters) and by simultaneous translocation of calcium through CTL plasma membrane with Ca2+ ionophores.2 Our results demon­strate that PMA/ A23187 very efficiently trig­ger BLT-E secretion of purified NK cells. In less than 2 hour incubation tirne almost half of the total BLT-E content is released from NK cells. On the other hand, when NK cells were incubated with K562 cells, BLT-E mea­surement reached stable values in 3-4 hours and, in that tirne less than one third of total BLT-E content was released. 
Because PMA/ A23187 triggering acts on CTL and NK cells, experiments should be done only with homogenous celi populations -CTL or NK celi clones or purified cells. On the other hand, K562 cells trigger NK celi exocytosis without influencing CTL exocyto­sis. 3 That would theoretically enable studies of NK celi exocytosis in mixed celi popula­tions (e.g. patients PBL). Our results, when using PBL in BLT-release test, demonstrate very low BLT-E release. A possible reason lies in calculation of BLT-E release: an enzyme release is expressed as a percentage of specif­ic release versus total enzyme celi content. Because a total celi enzyme content in PBL is a sum of total enzyme content in CTL and 
Radio/ 011col 1999; 33(1): 27-33. 
NK cells, NK celi release makes usually very low proportion of the sum. Furthermore, a calculation of BLT release can be greatly influenced by the proportions of CTL and NK cells in PBL. 
Obviously the experiments are best to be performed by using isolated NK cells to obtain high BLT-E release/effector celi. On the other hand, to perform extensive clinical studies, NK celi isolation may not be appro­priate method because considerable amounts of blood are required. As a compromise, we performed a simple NK-enrichment proce­dure by using Percoll gradient centrifugation. By using NK-enrichment procedure we were able to obtain PBL containing more than 45% of NK cells. Furthermore, to perform BLT-E release assay and NK-cytotoxicity test as well in most instances only 10 ml of blood was required i.e. the quantity that enables to per­form more extensive clinical studies. 
Our results confirm a close association between granule enzyme exocytosis and NK lytic process in healthy persons. A correla­tion between NK test and BLT-E release results increases with the purity of NK cells, used in the experiment. Because a granule enzyme exocytosis is supposed to be a part of a lytic process in NK cells, a BLT-E release assay could be used to further analyse the lytic process in NK cells. A described assay procedure is simple and requires only moder­ate blood volumes, so, it is possible to per­form it in clinical studies. Its use might be important especially in patients with impaired NK cytotoxicity (cancer patients, immunodeficient patients) because a defect of NK celi cytotoxicity can be influenced also by the factors that do not include granule enzyme exocytosis (NK celi binding and recognition of target cells, cytokine stimula­tion, activation of NK lytic machinery). 

llrn11 A / Natura! kil/er cel/s 
References 

1. 
Coleman PL, Green GOJ. A coupled photometric assay for plasminogen activator. Methods Ency1110/ 1981; 80: 408-14. 

2. 
Pasternack MS, Eisen HN. A novel serine esterase expressed by cytotoxic T lymphocytes. Nature 1985; 314: 743-5. 

3. 
Takayama H, Sitkovsky MV. Antigen receptor-reg­ulated exocytosis in cytotoxic T lymphocytes. J Exp Med 1987; 166: 725-43. 

4. 
Ciccone E, Viale O, Pende O, Ihan A, Oi Oonato C, Orengo AM, Biassoni R, Verdiani S, Moretta A, Moretta L. Involvement of HLA class I alleles in natura] killer celi specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2). J Exp Med 1992; 176: 963­72. 

5. 
Ihan A. Celi surfacc molecules that regulate abili­ty of NK cells to kili their targets. Zdrav Vest 1994; 63 suppl II: 53-5. 

6. 
Robertson MJ, Ritz J. Biology and clinical rele­vance of human natura] killcr cells. B/ood 1990; 76: 2421-38. 

7. 
Young JOE, Cohn ZA. Cell-mediated killing: A common mechanism? Cel/ 1986; 46: 641-2. 

8. 
Šmid L, Žargi M, Rok B, Kotnik V, Ihan A, Rode 


M. The rate of natura! killer cells and their cyto­toxic activity in patients with advanced pharyn­geal and laryngeal cancer. Radio/ 011col 1993; 27: 27-39. 


9. 
Ihan A, Kri_man 1, Ferlan-Marolt V, Tepe_ B, Gubina M. HLA-OR expression on CO8 lympho­cytes from gastric mucosa in urease-positive and urease-negative gastritis. FEMS [1111111111 Microbiol 1995; 10: 295-300. 

10. 
Nagatake H, Watanabe N, Kobayashi Y. Granule exocytosis in a cultured human natura] killer cell­rich population induced by K562 target cells and by PMA/ A23187. Cell f111111u110/ 1995; 161: 295-8. 

11. 
Bonema JO, Rivlin KA, Ting AT, Schoon RA, Abraham RT, Leibson PJ. Cytokine-enhanced NK cell-mediated cytotoxicity. Positive modulatory effects of IL-2 and IL-12 on stimulus-depcndent granule exocytosis. J l11111111110/ 1994; 152: 2098-104. 

12. 
Mittrucker HW, Fleischer B. Functional localisa­tion of an exocytosis-triggering C-protein in human cytotoxic T lymphocytes. /11111111110/ogy 1992; 76: 610-5. 

13. 
Bartels F, Bigalke H. Restoration of exocytosis aftcr inactivation of intracellular tetanus toxin. Illject f1lllmm 1992;60: 302-7. 

14. 
Trinchieri G. Biology of natura] killer cells. Adv /11111111110/ 1989; 47: 187-376. 

15. 
Moretta L. Ciccone E. Moretta A. Hoglund P. Ohlen C. Karre K Allorecognition by NK cells: nonself or no self?.111111111110/ Today 1992; 13: 300-6. 



Radio/ 011co/ 1999; 33(/): 27-33. 

Radio/ Oncol 1999; 33(1): 35-41. 
Micronuclei in cytokinesis-blocked lymphocytes of patients following iodine-131 radiotherapy 
Vilena Kašuba1, Zvonko Kusic,z and Vera Garaj-Vrhovac1 
1 

Mutagenesis Labaratary, Institute far Medica! Research and Occupatianal Health, 2 Department far Nuclear Medicine and Oncalagy, Clinical Haspital "Sestre milasrdnice" Zagreb, Croatia 
The micronucleus assay in cytochalasin-B cytokinesis blocked periphera/ blood lymphocytes in 10 patients with hyperthyreosis and various types oj thyroidal carcinomas was investigated. Patients received 259 ­5180 MBq I-131 sodium iodide perarally. Micranucleus (MN) frequencies wcre measured beforc and after 1-131 administration. Pre treatnzent MN rcsults were regarded as cantrols. Our results show considcrablc variability concerning agc and activity applied. A/so, thc number oj MN and thc numbcr oj binuc/eated cel/s with micronuc/ei (BNMN) showed Poisson regression adjust far within subject correlation overdispcr­sion. Log dose alonc was not significant. The interaction oj tiJne and dose was significant at higher dases,whilc thc rate oj MN changing was slowcr. Rclative risk time was calculated far the lowest dosc (259 MBq). By doubling the dose, the rate oj daily incrcase in lhe number oj MN and BNMN decreases by approximately 5% (Rclative risk MN =0.955; Relative riskBNMN = 0.954). 
Key words: hypcrthyroidism-radiotherapy; thyroid neoplasms-radiothcrapy; iodine radioisotopcs-advcrse effccts; lymphocytes; micronucleus test 
Introduction 
Radioiodines are often used for experimental purposes, diagnosis and therapy in clinical practice. The ionizing energy of radioiodine affects not only the thyroid with its uptake points, but other tissues as well, especially lymphocytes during their circulation through 
Received 20 July 1998 
Accepted 5 December 1998 Correspondence to: Vilena Kašuba, Mutagenesis Lab­oratory , Institute for Medica! Research and Occupa­tional Health, Ksaverska c. 2, P.O.Box 291, 10 000 Zagrab, Croatia. Tei: +385 1 467 31 88; Fax: +385 1 467 33 03; E-mail: vkasuba(i1
>imi.hr 

and around the gland containing the radioisotope. Therefore, it seemed to be of interest to investigate the cytogenetic alter­ations in blood lymphocytes of patients treat­ed with iodine-131. 
In order to determine the genotoxic risk associated with the diagnostic and therapeu­tic exposure to iodine-131, we conducted a follow-up study on the frequency of micronu­clei in cytochalasin-B blocked blood lympho­cytes. 

Until now the scoring of chromosome aberrations has been considered the most rel­evant method for the cytogenetic dosimetry. Nevertheless, in the last few years, the 

Kašuba Ve/ ni./ Micro11uclei i11 ly111phocyles after iodi11e-131 rndiothempy 
micronucleus assay in human peripheral blood lymphocyte cultures using the cytoki­nesis-block method has been demonstrated to be a fast and sensitive cytogenetic tech­nique and has been receiving increased atten­tion far biological monitoring of radiation 
1 2 

exposure., 
Counting micronuclei in cells which have undergone one cell division after the clasto­genic insult is deemed to be a simpler and more sensitive method than conventional chromosome aberration assay.3 Micronuclei originate from acentric fragments or from whole chromosome aberration assay and may serve as a measure far both chromosome 
3 

breakage and loss.1, 
The aim of this study was to identify the relation between the number and distribu­tion of micronuclei, using diagnostic and therapeutic doses of radioiodine-131. The fre­quency of micronuclei in cultured and cytoki­nesis-blocked circulating lymphocytes was determined befare the treatment and at sev­era! short intervals after it. The quantity of perorally administrated iodine-131 depends on what the clinical practice determines to be a sufficient therapeutical dose far either hyperthyroidism or thyroid carcinomas. The doses applied in our study ranged between 259 and 5180 MBq. 
Materials and methods 

Lymphocyte cultures and micronucleus test 
Venous blood was taken from 10 patients, 2 males and 8 females between 22 and 83 years of age, with Basedow-Graves disease (hyper­thyreosis) and thyroid carcinomas, treated with 1311 (Table 1). First we quantified the micronuclei in the samples taken befare treatment, and then we repeated the mea­surement on samples taken at short intervals (day 1,2 and 4) after treatment. Duplicate lymphocyte cultures in F-10 medium (Gibco, 
Radio/ 011col 1999; 33(1): 35-41. 


GB) supplemented with 20% of new-born calf serum (Biological Industries, Israel), penicillin (10 000 IU/ml) and streptomycin (10 000 µg/ml) were set up. Lymphocytes were stimulated by phytohaemagglutinin (PHA-Murex, GB). The cultures were incu­bated far 72 hours at 37 ° C, and at 44 h after the initiation of cultures cytochalasin-B (Sigma, Germany) dissolved in dimethyl­sulphoxide was added to arrest cytokinesis. The cells were collected by centrifugation, and instead of hypotonic treatment, we used 0.9% sodium chloride far 5 minutes at room temperature, fixation in methanol: glacial acetic acid, 3:1. Air dried slides were stained with 5% Giemsa solution. Each sample was analyzed only far binuclear cytochalasin B blocked cells with well-preserved cytoplasm. In each sample 1000 binucleated cells were analysed. The slides were scored at lO00x magnification. 
Statistica/ methods 
Far the statistical analysis we used Poisson regression adjusted far within subject corre­lation. The data were analysed using the SAS 612.PROC.GENMOD statistical package. 
Results 

Table 1 shows age, sex, diagnosis, the applied activity far each patient, the tata! number of micronuclei (MN) in cytochalasin B cytokine­sis blocked peripheral human blood lympho­cytes in 1000 binucleated cells, and the num­ber of binucleated cells with micronuclei (BNMN) in patients befare and after radioio­dine application, in order to show the MN distribution among cells. In the group stud­ied, there was a considerable variability con­cerning age and activity applied. 
In some cases the cytogenetic study was not able to be carried out in all samples due to low stimulation of the cultured lympho­
F 
1 53 Hyperthyreosis 56 45 78 64 81 68 119 100 
F 

84 72 62 
2 F 29 Hyperthyreosis 262 24 18 
3 
;,,: "' 
.f 
g­sc:: . 
Table l. Dose, age, sex, diagnosis, total number of micronuclei (MN) in 1000 binucleated cells, number of binucleated cells with micronuclei (BNMN) in the 
hyperthyroidism and carcinoma patients treated with radioiodine-131 
=, 
:---
Aplicated Post-treatment Post-treatment Post-treatment 
" 
Subject Sex Age Diagnosis activity Pretrea tmen t (24 hours) (48 hours) (96 hours) s: 
;::;· 
(years) (MBq) MN BNMN MN BNMN MN BNMN MN BNMN 
5.. .-. 
56 Hyperthyreosis 309 26 24 60 100 91 
'f 
--;:; 

4 
F 
40 Toxic adenoma glandulaae thyroideae 1176 22 22 34 31 44 
­
5 F 57 Papillary thyroid carcinoma 2960 22 18 28 23 32 29 32 30 
6 
F 
67 Follicular thyroid carcinoma 2960 22 19 52 46 83 
s
7 F 83 Papillary thyroid carcinoma 2960 70 56 69 57 62 53 . 
8 M 22 Mixed follicular/papillary carcinoma 3700 20 16 15 14 20 16 25 
metastases 9 F 64 Papilary thyroid carcinoma 3700 25 24 12 12 29 26 19 17 
2 
10 M 71 Follicular thyroid carcinoma -distant 5180 102 82 156 130 100 84 117 98 
"" 
C, 
Al "' metastases s:. 
­
"" 
2 
2.. ""' 
"' 
9 
" 
2.. 
,_., 
<,o 
<,o ,'-? 
w 
w 
y, . (;.) "l 

Kašulm Vel al. j Micronuc/ei in lymplzocytes after iodine-131 radiotherapy 
Table2. Results of statistical analysis 

Variable  Parameter  Relative risk  Lower confidence limit  Upper confidence limit  p  
1  MN tirne (day)  1.27494  1.04487  1.55566  0.0000  
2  MN tirne x dose  0.95530  0.93518  0.97585  0.0000  
3  MN age (10 years)  1.22708  1.09297  1.37763  0.0005  
4  BNMN tirne (day)  1.29153  1.05895  1.57520  
5  BNMN tirne x dose  0.95426  0.93418  0.97477  
6  BNMN age (10 years)  1.21817  1.08966  1.36184  

cytes. We observed binucleated cells with 1-4 micronuclei (Figure 1) 
Poisson regression adjusted for within subject autocorrelation was used to compare the variation in the frequency of MN and BNMN in the four sampling times. The results in Table 2 show that the number of micronuclei increased in tirne. For both MN and BNMN the models showed overdisper­sion. For MN the deviance was 473.5 and Pearson x2 515.7 with 33 degrees of freedom. For BNMN the deviance was 364.5 and Pear­son,s x2 was 399.5 with 33 degrees of free­dom. We have introduced a scaled in the fina! model to adjust for overdispersion (for MN scale was 3.7878 and for BNMN 3.3234). 
Log dose alone was not significant. The interaction of tirne and dose was significant at higher doses, the rate of the MN changing was slower. A relative risk for tirne was calcu­lated for the lowest dose (259 MBq). By dou­bling the dose, the rate of daily increase in the growth of number of MN and BNMN decreased for about 5% (Relative 
= 
riskMN =0.955; Relative riskBNMN 0.954). 
The data obtained when patients were classified into five subgroups, according to the activity received ( < 300 MBq, 1176 MBq, 2960 MBq, 3700 MBq, 5180 MBq) are shown graphically in Figure 2. These values show wide variability in the baseline frequency of BNMN among patients before treatment. 
140 120  --+.-<300 MBq ---lt,--1176MBq --2960MBq .3700M8q ;+; 5180 MBq  
100  .  /  

v . 
80 
z 
z 
60 
40 
20 
0-f------------­
Pre-24 h Post-48 h Post-96 h Post-treatment treatment treatment treatment 
Sampting tirne (hours) 
Figure 2. Frequency of BNMN before and after the treat­ment. 
Discussion 
A consequence of radiation-induced DNA damage is the formation of micronuclei, which appear similar to the main nuclei but are smaller and have a reduced DNA content.4 
The use of micronucleus assay to evaluate radiation exposures resulting from internally deposited radioactive materials in people pre-
Radio/ Onco/ 1999; 33(1): 35-41. 

Kašuba Vet al. / Micronuclei in lymphocytes after iodine-131 rndiotherapy 

4 
C 
• 


d 

Figure l. Photomicrograph of binucleate human blood lymphocyte without micronuclei (a) , with one micronu­clei, (b), with two micronuclei (c) and with four micronu­clei (d). Stained by 5% Giemsa (magnification 1000 x) 
sents severa! specific problems. First, the deposition, distribution, and dose to individ­ual cells are dependent on the radionuclides involved, the route of exposure, the metabol­ic state of the individual and the chemical and physical form of the material. Second, because of individual differences, it is impos­sible to make a reliable estimate of the radia­tion dose that the individual receives even if the exposure leve!, radionuclides involved, and their physical and chemical form are known. 
For nonuniformly distributed internally deposited radionuclides, it is important to recognize that not only the radiation dose is nonuniform, but also the lymphocytes used to evaluate the exposure are nonuniformly distributed in blood, organs, lymph nodes and lymph follicles throughout the body. 
It is well established that there is hetero­genity in the repair of lesions along the DNA molecule.5-8 The level of gene activity and repair at the level of the chromosome are crit­ical factors that may influence the formation of chromosome aberrations and micronu­clei.9 
The obtained results indicate that the var­ied cell response to low doses depends on individual features of the patients, quite simi­lar to those observed by Brown.10 Moreover, it is important to notice a corresponding irregularity in the distribution of the incorpo­rated radionuclides, not only in the doses, but in the lymphocytes irregularly distributed in blood, organs, lymph nodes and follicles. The obtained results as well as the referred 
11,12 , 13 
literature point out individual differ­ences in reacting to exposure. As there is variation in the proliferation rate of the lym­phocytes from different individuals, kinetic differences appear to remain a source of vari-
Radiol Oncol 1999; 33(1): 35-41. 

Kašuba Vel al. / Micronuclei in lymphocyles after iodine-131 radiotherapy 
ability in the MN assay.14 Keldsen and his coworkers15 noticed considerable absorption differences between patients equally exposed to iodine-131 (here patients 6, 7 and 9). It is interesting to note that some patients have micronuclei control values within the range of those seen in healthy population.'16 This finding has been confirmed by the results of our study (patients 1, 2, 3, 6, 7, 8, and 10). It seems that the loss of lymphocytes is associ­ated with big lesions. 
The evaluation of damage immediately after the exposure is incomplete. The reason can be sought in the temporary mitotic delay as well as in the potential transfer of chromo­somal instability from the exposed parental to daughter cells.'17 The comparison of ali patients in the study brings farward big indi­vidual differences since the changes were noted befare the exposure (patients 1, 7, and 10). Unable to determine the etiology of such changes, we came to the conclusion that the results obtained after the therapeutic doses of radiopharmaceuticals should be evaluated with respect to the results obtained befare the exposure. 
If we want to compare our results with the similar study of Gutierrez and coworkers,2 it is evident that in their group of subjects there is a significant increase of 5% of MN per week, compared to pre-treatment, and after that MN is lower than in the pre-treatment period. The rate of increase was essentially lower than in our results. In the study by Gutierrez et al., a tirne change was signifi­cant, but one could not observe the interac­tion between tirne and dose. Log dose and group, together, were significant. 
Depending on age, our results point out an increase in MN by 20% far ten years. 
Radiotherapy treatments are very toxic far lymphocytes, and could result in multilocus mutagenesis which could affect cloning effi­ciency of „hit" cells. Seifert et al.11 calculated that each lymphocyte of a radiotherapy treat­ed patient carried an average of six induced 
Radio/ Oncol 1999; 33(1): 35-41. 
mutations. This gJ·oup also observed a large individual variation in the reaction exposure. One of the causes of interindividual variation could be the polymorphism at the large num­ber of loci involved in repair of DNA damage.18 Another one is individual body size, which influences the dose to the target tissues. Finally, the differences in each indi­vidual's physical or chemical environment may be involved in the heterogeneity of response. 
Acknowledgment 

The author acknowledges the cooperation with Mr.Sc. Diana Šimic, from the Depart­ment of Biomathematics at the Institute far Medica! Research and Occupational Resear­ch. 
References 
1. 
Fenech M. The cytokinesis-block micronucleus technique: A detailed description of the method and its application to genotoxicity studies in human populations. Mulat Res 1993; 285: 35-4. 

2. 
Gutierrez S, Carbonell E, Galorfe P, Creus A, Mar­cos R. Micronuclei induction by 131 1 exposure: Study in hyperthyroidism patients. Mulat Res 1997; 373: 39-45. 

3. 
Fenech M, Morley AA. Measurement of micronu­clei in lymphocytes. Mulat Res 1985; 147: 347-67. 

4. 
Schiffmann D, De Boni U. Dislocation of chro­matin elements in prophase induced by diethyl­stilbestrol: a novel mechanism by which micronu­clei can arise. Mulat Res 1991; 246: 113-22. 

5. 
Keyse SM, Tyrell RM. Rapidly occurring DNA excision repair events determine the biological expression of u.v.-induced damage in human cells. Carci11oge11esis 1987; 8: 1251-6. 

6. 
Sapora O, Belli M, Maione B, Pazzaglia S, Taboc­chini MA. The influence of genome structural organization on DNA damage and repah-in eucaryotic cells exposed to ionizing radiation. In: Fielden E M and O Neil P (eds) The Earl Effects oj Radia/ion 011 DNA. Springer-Verlag, Berlin; 1991.p. 85-101. 





Kašuba Vet al. / Micro1111clei i11 lymphocytes after iodine-131 radiotherapy 
7. 
Larrninat F, Zhen W, Bohr VA. Gene-specific DNA repair of interstrand cross-links induced by chernotherapeutic agents can be preferential. J Biol Chem 1993; 268: 2649-54. 

8. 
Cooper PK. Defective repair of ionizing radiation darnage in Cockayne syndrorne and XP-G. Forty­second Annual Meeting oj the Radiatio11 Researc/1 Society. 1994. A/Jslracls S24-2. Radiation Research Society, !L. 

9. 
Walker J-A, Boreharn DR, Unrau P, Duncan AMV. Chrornosorne content and ultrastructure of radia­tion-induced rnicronuclei. Mutageuesis 1996; 11: 419-24. 

10. 
Brown RA, Gibbs JH, Robertson AJ, Potts RC, Beck SJ. Development of asynchrony in growth of normal human lyrnphocytes during first day in culture after PHA stimulation. Exp Celi Res 1980; 126: 87-97. 

11. 
Seifert AM, Bradley WEC, Messing K. Exposure of nuclear medicine patients to ionizing radiation is associated with rises in HPRT-mutant frequency in peripheral T-lymphocytes. Radia/ Res 1987; 191: 57-63. 

12. 
Huber R, Braselmann H, Bauchinger M. lntra-and 



inter-individual variation of background and radi­
ation-induced micronucleus frequencies in human 
lymphocytes. In/ J Radio/ Biol 1992; 61: 655-61. 

13. 
Pcrera FP, Whyatt RH .Biomarkers and molecular epiderniology in mutation / cancer research. Mulat Res 1994; 313: 117-29. 

14. 
Falck G, Catalan J, Norpa H. Influence of culture tirne on the frcqucncy and contents of human lymphocyte micronuclei with and without cytochalasin B. Mulat Res 1997; 392: 71-9. 

15. 
Keldsen N, Mortesen BT, Hansen HS. Hematolog­ical effects frorn radioiodine treatrnent of thyroid carcinorna. Acto Onco/ 1990; 29: 1035-9. 

16. 
Gantenberg H-W, Wutke K, Strefer C, Muller W­


U. Micronuclei in human lymphocytes irradiated in vitro or in vivo. Radio/ Res 1991; 128: 276-81. 

17. 
Galloway SM. Chromosome aberrations induced in vitro: rnechanisrns, delayed expression, and intriguing questions. Environ Mol Mutagen 1994; 23 (Suppl.24): 44-53. 

18. 
Herrllich P, Mallick U, Ponta H, Rahrnsdorf HJ. Genetic changcs in marnmalian cells reminiscent of an SOS response. Human Genet 1984; 67: 360-8. 


Radio/ Oncol 1999; 33(1): 35-41. 

Radio/ 011co/ 1999; 33(1): 43-53. 
Prognostic relevance of urokinase plasminogen activator and its inhibitors in patients with breast cancer 
Simona Borštnar, Tanja Cufer, Ivan Vrhovec, Zvonimir Rudolf 
Institute oj Oncology, Ljubljana, Slovenia 


Urokinase plasminogen activator (uPA) and its inhibitors, PAI-1 and PAI-2, play an important role in inter­cellular tissue degradation, thus promoting tumor cel/ invasion into the adjoining sh'uctures and metastasiz­ing. Our study was aimed to assess a possible prognostic valuc oj uPA, PAl-1 and PAI-2 in a retrospective series oj 87 patients with breast canccr stage 1-IIJ, whosc cytoso/s were stored in thc arclzivcs oj thc Insti­tute oj Oncology in Ljubljana. The median jollow-up was 35 months. The prognostic valuc oj the estab­lished prognostic jactors and uPA, PAl-1 and PAl-2 were evaluated by means oj univariate statistica/ analy­sis and partial multivariafe mode/s. The obtained uPA values were very low and did not correlate with thc disease-jree survival, whereas PAI-1 and PAI-2 signijicantly injluenced the timc to tlze Jirst recurrence. Patients with PAl-1 values above 5 ng/mg proteins had statistically signijicantly worse disease-Jree sur­vival than the patients with Iower PAI-1 values (58% vs. 85%). In the case oj PAl-2, thc situation was just the opposite: the patienfs with PAl-2 values exceeding 6.4 ng/mg proteins had statistically signijicantly bet­ter 3-year diseasc-jrec survival than the patients with lower values (90% vs. 60%). Both, PAl-1 and PAl-2 retained their independent prognostic va/ue, irrespective oj thc addition oj the estab/ished prognostic jac­tors to partial multivariate mode/s, and only with /ocally advanced disease the prognostic value oj PAI-1 was greater than that oj PAI-2. 
Key words: breast neoplasms; urokinase; plasminogen activator inhibitor 1; plasminigen activator inhibitor 2; prognosis 
Introduction 

A number of extracellular proteolytic enzymes are expressed in the tumor tissue; these are involved in the invasion of tumor cells into the surrounding tissues as well as 
Received 31 August 1998 
Accepted 10 December 1998 
Correspondence to: Simona Borštnar MSc, Institute of 
Oncology, Zaloška 2, S1-1000 Ljubljana, Slovenia. 
in distant dissemination process. The central role among proteolytic enzymes is attributed to the serine proteinase -urokinase plas­minogen activator (uPA) and to uPA 
1,2,3 inhibitors types 1 and 2 (PAI-1 and PAI-2).

A decade ago, researchers came to the idea that serine proteinases could be an indicator of the metastatic potential of tumors, and thus also a prognostic factor of cancer. In the last decade, a number of studies were carried out which were aimed to assess the prognos­
Bošt11ar Seta/./ Urokinase plasmin oge11 activator an d its inhibitors 
tic relevance of uPA, PAI-1 and PAI-2 in breast cancer and other solid tumors.4 
In his first study concerned with the prog­nostic value of serine proteinases, Duffy and co-workers determined uPA content in the tumor cytosols from patients with breast can­cer, which were prepared for routine determi­nation of hormone receptors.5 The results of this study for the first time established a cor­relation between uPA and the prognosis in breast cancer patients. Severa! other authors later on also confirmed the same findings.6-9 From the 90's on, a similar relevance was reported for PAI-1 determined in the cytosols from breast cancer patients_rn-14 The mea­sured mean and cut-off values of uPA and PAI-1 differed from one study to another. The reason for this variability could be attributed to different tumor tissue preparation tech­niques and different buffers used. Because of those differences, a standard cut-off value that would delineate high values from low ones could not be determined. However, the results of investigations performed so far unequivocally speak in favor of the indepen­dent prognostic value of uPA and PAI-1.6-14 High values of either of these two factors are associated with a higher risk of recurrence and a shorter survival. PAI-2 has not been sufficiently investigated yet. For the differ­ence from uPA and PAI-1, high PAI-2 values were found to be associated with a favorable prognosis.9•10 So far, the independent prog­nostic value of PAI-2 has been confirmed by one of the two studies performed.9 
Our retrospective study was aimed to established whether the values of uPA, PAI-1 and PAI-2 determined in tumor cytosols pre­pared with phosphate buffer, which are oth­erwise used routinely for hormone receptor determination, correlate with the established prognostic factors, and whether they signifi­cantly influence the disease free survival of patients with breast cancer. 
Materials and methods 
Our retrospective study was carried out in a series of 87 patients with operable and locally advancer breast cancer, who were admitted to the Institute of Oncology for the first time in 1994 or in the first two months of 1995, and operated on for cytologically confirmed breast cancer. 
Deep-frozen tumor cytosols from those pa­tients are stored in the cytosol bank of the Insti­tute of Oncology in Ljubljana. The prepared cytosols are kept at a temperature of minus 20°C. The cooling was never discontinued. 
Patients, tumors and treatment characteristics 
The data on age, menopausal status, clinical tumor size, clinical lymph node status, patho­logical tumor size, pathohistological tumor type, malignancy grade, axillary lymph node involvement, hormona! receptor status, and primary treatment were derived from the patient record files stored in the archives of Institute of Oncology. 
The stage of disease was classified accord­ing to UICC-WHO criteria (UICC, 1974). While the latter criteria were used for patho­histological tumor type determination, the grade of malignancy was assessed according to Scarf-Bloom-Richardson' s classification.10 The cut-off limit for positive estrogen and progesterone receptors was set at value > 10 fmol/mg proteins. 
Ali the patients underwent a local radical treatment. Ali of them also had axillary lyrn­phadenectorny perforrned. In the case that only conservative surgery was feasible, the patients were additionally irradiated to the area of the operated breast. Patients with positive axillary lyrnph nodes received adju­vant chernotherapy. The same was also given to the patients with negative axillary lyrnph nodes and presence of the established unfa­vorable prognostic factors (e.g. large and/or poorly differentiated tumor). 
Radio/ On col 1999; 33(1): 43-53. 

Boštnar Set al. / Urokinase plasminogen activator and its inhibitors 
The mean age of patients at diagnosis was 52 years (range 29-75 years). Other character­istics of the patients and tumors are present­ed in Table l. 
Tissue preparation technique and the deterrnina­tion oj urokinase system components 
Immediately upon surgery, the removed tumor tissue was stored in liquid nitrogen. In the process of cytosol preparation, the frozen tumor was first ground with a microdysmem­brator. The obtained powder was suspended in phosphate buffer (5 mM Na2HPO4, 1.7 mM KH2PO 4, 1 mM monothioglycerol, 10% (v/v) glycerol, pH 7.4), and the suspension ultra-centrifuged at 100,000 x g for 45 min, at 
The total uPA concentration in the cytosol and extract was determined with IMUBIND® Tissue uPA ELISA Kit, while PAI-1 was deter­mined with IMUBIND® Tissue PAI-1 ELISA Kit, and PAI-2 with IMUBIND® Tissue PAI-2 ELISA Kit (American Diagnostica Inc.) 
Follow up 
After completed primary therapy, the patients were subjected to regular follow-up examinations at the Institute of Oncology. The data on possible tirne and site of progres­sion were derived from patients records. 
The patients were followed up for 1-49 months (median follow up was 35 months). 
Data processing 
Interdependence of the urokinase system components with other primary tumor char­acteristics was determined on the basis of contingency tables and chi-square test. The influence of the component of urokinase system on the disease-free survival was pre­sented by means of Kaplan-Meier' s survival curves, and any differences in the survival analyzed with the log-rank test.15, 16 The mul­tivariate regression analysis by Cox was used for the evaluation of independent prog­nostic value of urokinase system compo­nents.17 Statistical analysis and graphic pre­sentation of the results were done using ,,Statistica for Windows" and „BMDP" pro­gram packages. 
Results 

Urokinase system measurements 
In 87 patients, the range of uPA values (con­centrations) in the cytosol prepared with phosphate buffer was 0-1.83 ng/mg proteins, median 0.34 ng/mg proteins, the lower and the upper quarts being 0.15 and 0.53 ng/mg proteins, respectively. 
In the same series of 87 patients, the range of PAI-1 levels in the cytosol prepared with phosphate buffer was 0.06-75.91 ng/mg pro­teins, median 6.02 ng/mg proteins, while the lower and the upper quarts were 3.77 and 
8.93 ng/mg proteins, respectively. 
The range of PAI-2 values in the cytosols prepared with phosphate buffer was 0.31­
75.80 ng/mg proteins, median 3.35 ng/mg proteins, the lower and the upper quarts being 1.51 and 12.31 ng/mg proteins, respec­tively. 
The influence of urokinase system components on disease-free survival 
Within the median observation period of 35 months, the disease was found to recur in 28/87 patients (32%). Three patients (11 %) presented with local recurrence, while 19 patients (68%) had distant metastases alone, and 6 patients (21 %) had both. Three-year disease free survival of the whole group of patients was 69%. Disease free survival for the whole group of 87 patients is presented in Figure l. We were trying to determine the cut-off values of uPA, PAI-1 and PAI-2, which 
Radio/ Oncol 1999; 33(1): 43-53. 

Boštnar Set al. / Urokinase plasminogen activator and its inhibitors 
Table 1. Charaeteristies of 87 patients 

Number  (%)  
Patients  Menopausal status premenopausal postmenopausal unknow  29 57 1  33 66 1  
Tumor size  
T1  8  9  
T2  54  62  
T3  13  15  
T4  12  14  
Nodal status  
NO  54  62  
Nl  26  30  
N2  7  8  
Stage (UICC-lnternational Union against Caneer) I  8  9  
II  60  69  
III  19  22  
Tumors  Pathologieal tumor size Tpl Tp2 Tp3 Tp4 unknown  1 13 57 14 2  1 15 66 16 2  
Pathohistologieal tumor type invasive dueta!  76  88  
invasive lobular  8  9  
mucinous  1  1  
others  1  1  
unknown  1  1  
Differentiation grade (invasive dueta] eareinoma) GI  4  5  
GII  21  28  
GIII  48  63  
unknown  3  4  
Number of positive nodes o  30  34  
1-3  26  30  
>3  31  36  
Estrogen reeeptors .10 ng/mg protein >10 ng/mg protein Progesterone reeeptors .10 ng/mg protein _____:>_10 ng/mg protein  43 44 60 27  49 51 69 31  

Radio/ Oncol 1999; 33(1): 43-53. 

Boštnar Seta/./ Ll rokinase plas111in oge11 activator an d its i11/zi/Jitors 
0.9 ] C 0.8 
5; 
0.7 ] & 0.6 
.. 05 
Q 0.4 
i' 
0.3­1 0.2 P-. 0.1 
12 18 24 30 36 42 48 
Time (months) 

Figure l. Relapse-free survival of 87 breast cancer patients. 
could best differentiate patients with favor­able prognosis from those with unfavorable one. 
It has been found that the measured uPA values in the cytosols from our series of patients failed to correlate with the disease­free survival. The cut-off value of PAI-1 in our series of patients was 5 nglmg proteins. The disease-free survival of patients with PAI-1 values exceeding 5 ng/mg proteins was statis­tically significantly worse than that of the patients with PAI-1 values under 5 nglmg proteins (P = 0.0046) (Figure 2). 

Time (months) 

Figure 2. Rclapse-free survival according to PAl-1. 
The cut-off value of PAI-2 in our series of patients was 6.4 nglmg proteins. The disease­free survival of patients with PAI-2 values 
1.of=.====..-...-..­
o.9­

ro 
> ·E 0.13 
. 0.7 
0.6-1 & 05 '6 
0 0.4 
!F0.017S 

g' 0.3 
..o 0.2­
P-. 0.1 
a1=.-.. 
0-1 =.!1J.j..·..t:"_,,::_::::.=.J..--11 .-----.­
­
12 18 24 30 36 42 
Time (1nonths) 

Figure 3. Relapse-frees urvival according to PAI-2. 
exceeding 6.4 nglmg proteins was statistical­ly significantly better that that of the patients with PAI-2 values under 6.4 nglmg proteins (p = 0.0178) (Figure 3). 
Comparison oj the influence oj different prognos­tic factors on disease-free survival 
Univariate analysis (log-rank test): The influ­ence of menopausal status, clinical tumor size, clinical lymph node status, stage of the disease, pathohistological tumor size and grade of malignancy grade, pathohistological evidence of axillary lymph node involvement, presence of estrogen and progesterone recep­tors, as well as PAI-1 and PAI-2 content in the tumor on disease free survival was studied. It was found that disease-free survival was sig­nificantly influenced by 7 /11 factors under study. The influence of clinical tumor size (operable cancers -T2 and T3 vs. locally advanced disease -T4) was also statistically significant. In our series, the patients with operable cancers presented with 73% 3-year survival, while those with locally advanced disease had only 20% 3-year survival rate (p<0.00001). Eight patients with tumors smaller than 2 cm (stage Tl) were not includ­ed in the analysis. 
Stage of disease was another prognostic factor that turned out to be statistically sig­nificant for the disease free survival. In our 
Radio/ Oncol 1999; 33(1): 43-53. 

Boštnar Set al. / Urokinase plasminogen activator and its inhibitors 
series, 78% of patients with stage II at the tirne of diagnosis survived 3 years without evidence of disease, while only 32% of those with stage III were free of recurrence after 3 years (p<0.00001). Patients with stage I were too few (N=S) to be included into the statisti­cal analysis. Both clinical and pathological status of the axillary lymph nodes exerted statistically significant influence on the dis­ease free survival. While patients with non­palpable axillary lymph nodes survived 3 years without evidence of disease in 85%, those with palpable lymph nodes had only 45% 3-year disease-free survival (p<0.00001). After three years, the disease recurred in 42% of patients with pathologically positive axil­lary lymph nodes and in only 11 % of those with negative pathological lymph node find­ings (p=0.0053). The number of involved lymph nodes was also statistically significant. Thus, the patients with 1-3 positive axillary lymph nodes had 80% disease-free 3 year sur­vival, while in those with more than three positive lymph nodes this rate was only 41 % (p<0.00001). Further, the recurrence of dis­ease was significantly influenced by the con­tent (presence) of estrogen receptors in the tumor. Patients with negative estrogen recep­tors had lower 3-year disease-free survival than those with positive estrogen receptors, i.e. 60% vs. 80% respectively (p=0.0409). 
Both, PAI-1 and PAI-2 contents in the tumor significantly influenced the patients' survival. It turned out that the patients with PAI-1 tumor content exceeding 5 ng/mg pro­teins had statistically significantly worse dis­ease-free survival than the rest of patients under study (p=0.0046). Thus, the disease recurred within 3 years in 42% of patients with PAI-1 content above the cut-off value, and in only 15% of those with PAI-1 content below the cut-off value (Figure 2). 
With PAI-2, however, the situation was just the opposite. Patients with PAI-2 tumor content exceeding 6.4 nglmg proteins had statistically significantly better disease-free survival than the rest of the patients under study (p=0.0178). Thus, 10% of patients with PAI-2 values >6.4 ng/mg proteins presented with recurrence within 3 years, as compared to the patients with PAI-2 values below 6.4 ng/mg proteins in whom the recurrence rate was as high as 52% (Figure 3). The results of univariate analysis are presented in Table 2. 
Multivariate analysis: Independent prog­nostic value of PAI-1 and PAI-2 was studied by the multivariate Cox's regression model. Due to insufficient number of patients, we did not include into the model ali the seven factors that had shown their prognostic value in the univariate analysis; instead, we made a few partial multivariate models. Thus the remaining five factors shown as statistically relevant by univariate analysis were added one by one to the basic two factors studied (PAI-1 and PAI-2) (Table 3). 
When both inhibitors of plasminogen acti­vator alone were included into the multivari­ate model, PAI-1 and PAI-2 turned out to be strong prognostic factors, PAI-1 being the more relevant of the two. If only these two factors were considered, the relative risk of recurrence would increase by 5.9-times in patients with PAI-1 exceeding 5 ng/mg pro­teins, and by 4.4-times in those with PAI-2 values below 6.4 nglmg proteins. 
PAI-1 and PAI-2 did not loose their prog­nostic value by inclusion of other prognostic factors into the model. A stronger prognostic value was established only for tumor size (operable vs. locally advanced cancers) and stage (stage II vs. stage III). Both inhibitors of plasminogen activator were shown to have a stronger prognostic value that clinical and pathological status of the axillary lymph nodes. In the multivariate model with PAI-1 and PAI-2, estrogen receptors !ost their prog­nostic value. 
Radio/ Onco/ 1999; 33(1): 43-53. 

Boštnar Set al. / Urokinase plasminogen activator and its in/zibitors 
Table 2. Univariante analysis of disease free survival (log-rank test ) 
Prognostic factor  Number  Number of relapses  p  
premenopausal  29  10 (34)  0.8306  
postmenopausal  56  18 (32)  
Tumor size*  
T2+T3**  67  20 (30)  <0.0001  
T4  12  8 (67)  
Clinical nodal status  
palpable lymph nodes  54  9 (17)  <0.0001  
nonpalpable lymph node  33  19 (58)  
Stage*'"*  
II  60  14 (23)  <0.0001  
lII  19  14 (74)  
Pathological tumor size  
<20mm  14  3 (21)  0.2692  
.20mm  71  24 (34)  
Diferentiation grade****  
II  21  4 (14)  0.0980  
llI  48  19 (40)  
Pathological nodal status  
negative lymph nodes  30  5 (17)  0.0053  
positive lymph nodes  57  23 (40)  
Estrogene receptors  
SlO fmol/mg protein  43  18 (42)  0.0409  
<10 fmol/mg protein  44  10 (23)  
Progesterone receptors  
SlO fmol/mg protein  60  23 (38)  0.0978  
> 10 fmol/mg protein  27  5 (19)  
PAI-1  
<5 nglmg protein  35  5 (14)  0.0046  
>5 nglmg protein  52  23 (44)  
PAI-2  
<6.4 nglrng protein  58  24 (41)  0.0178  
>6.4 nglrng protein  29  4 (14)  

* 8 patients with tumor size <2cm (Tl) were excluded fron1 the analisis because the nun1bcr was to small for statistka] evaluation ** Difference between T2 and T3 was not statistically significant (log rank: p=0.1254) *** 8 patients with stage I werc cxcluded from the analysis becausc thc number was to small for statistical evaluation **** invasive dueta! carcinoma; 4 patients with grade I were cxcluded from the annlysis because thc number was to small for sta­tistical evaluation 
Discussion 

Our retrospective study was undertaken with the aim to show whether the components of urokinase system measured in tumor cytosols prepared with phosphate buffer for biochemical hormone determination had any prognostic value. 
Our retrospective study has failed to show any correlation between uPA values and dis­ease-free survival. Contrary to our results, the findings of three published retrospective 
Radio/ Oncol I999; 33(1): 43-53. 

Boštnar Set al. / Uroki11ase plasminogen activator and its inlzibitors 
Table 3. Multivariate Cox regression analysis 

Prognostic factor RR* 95% CI** p 
PAI-1 (<5 nglmg protein vs. >5 ng /mg protein) >5 ngl mg protein 5.89 2.21-15.66 0.0004 PAI-2 (>6.4 nglmg protein vs. < 6.4 ng /mg protein) 
<6.4 
ng /mg protein 4.26 1.47 -7.14 0.0079 PAI-1 (<5 nglmg protein vs. >5 ng /mg protein) >5 ngl mg protein 7.79 2.83 -21.46 0.0001 PAI-2 (>6.4 nglmg protein vs. < 6.4 ngl mg protein) 

<6.4 
ng /mg protein 3.57 1.20 -10.00 0.0218 Clinical tumor size 


(T2+T3 vs. T4)  
T4 8.34  3.39 -20.48  <0.00001  
PAI-1  
(<5 nglmg protein vs. >5 ngl mg protein)  
>5 ng /mg protein  5.65  2.83 -21.46  0.0006  
PAI-2  
(>6.4 nglmg protein vs. < 6.4 ng /mg protein)  
<6.4 ng mg/protein  4  1.41 -12.5  0.0100  
Clinical nodal status  
(palpable lymph nodes vs. nonpalpable lymph nodes)  
nonpalpable lymph nodes  3.75  1.67 -8.43  0.0014  
PAI-1  
(<5 nglmg protein vs. >5 ngl mg protein)  
>5 ng mg/protein  5.77  2.13 -15.63  0.0006  
PAI-2  
(>6.4 nglmg protein vs. < 6.4 ng /mg protein)  
<6.4 ng /mg protein  5.56  1.92-16.67  0.0018  
Stage  
(II VS. III)  
III  6.96  3.14 -15.40  <0.00001  

PAI-1  
(<5 nglmg protein vs. >5 ngl mg protein)  
>5 ng /mg protein  5.25  1.95 -14.12  0.0010  
PAI-2  
(>6.4 nglmg protein vs. < 6.4 ng /mg protein)  
<6.4 ngl mg protein  5.26  1.75 -14.29  0.0028  
Pathological nodal status  
(negative vs. positive)  
positive  3.81  1.39 -10.44  0.0092  
PAI-1  
(<5 nglmg protein vs. >5 ng /mg protein)  
>5 ng /mg protein  5.68  2.13 -15.13  0.0005  
PAI-2  
(>6.4 nglmg protein vs. < 6.4 ngl mg protein)  
<6.4 ng /mg protein  3.85  1.33 -11.11  0.0128  
ER  
( 10 fmol/mg protein vs. > 10 fmol/mg protein)  
> 10 fmol/mg protein  
 
 n.s.***  

* Relative risk; ** confidence interval; ***not significant 
Radio/ Onco/ 1999; 33(1): 43-53. 

Bošt11ar Set al. / Llrokinase plasmi11oge11 activator a11d its inlzibitors 


studies ascribe some prognostic value to uPA 
18 19 

measured in cytosols.11, , The authors 
obtained different median values (0.4 -0.52 nglmg proteins) and different maximum val­ues (3.2 -4.4 nglmg proteins), which were invariably higher than the values measured in cytosols during our retrospective study (median 0.34 nglmg proteins, range 0-1.83 nglmg proteins). This discrepancy could be explained by the fact that the cytosols from the archives of the Institute of Oncology were prepared with phosphate buffer alone, whereas the cytosols used in the three retro­spective studies reported were prepared with buffers and addition of EDTA. It seems that the latter substance improves the extraction of proteins, and associated with that uPA, to such an extent that the measurements become more reliable and the analytical error lesser. Apparently, the uPA values deter­mined in the tissue that has been processed according to the technique described are just high enough to allow for the determination of cut-off value which groups breast cancer 
patients by prognosis. 
For the difference from uPA, PAI-1 and PAI-2 in our study correlated with disease­free survival. The univariate analysis has shown a statistically significant influence of PAI-1 and PAI-2 content in tumor cytosols on disease-free survival. Patients with higher PAI-1 values presented with recurrence more frequently than those with lower PAI-1 values. With PAI-2 the situation was just the oppo­site: the recurrence rates within three years in patients with higher PAI-2 values were lower. A similar influence of PAI-1 on disease-free survival was also established by univariate analysis in some other studies investigating 
79 nt4 2o 
the prognostic value of PAI-1. 6, , ,, , Also 
the results of both studies on the prognostic value of PAI-2 are more or less consistent with 
10 
our study.9 , While the French study -like­wise ours -has confinned the association of high PAI-2 levels with a favorable prognosis, Foeckens et al. in their study on 1012 patients failed to confirm a correlation between PAI-2 values and disease-free survival or overall sur­vival. However, when their patients were grouped according to uPA tumor content, the patients with higher uPA values also had the cut-off value of PAI-2 determined, which dis­tinguished the patients by prognosis. It has been found that the patients with higher uPA content had a better prognosis if they also had high PAI-2 values.10 

Apart from PAI-1 and PAI-2, in our group of patients a statistically significant influence on the disease-free survival was also exerted by the established prognostic factors: clinical tumor size, clinical lymph node status, stage of the disease, pathological evidence of axil­lary lymph node involvement, and the pres­ence of estrogen receptors in the tumor. In the evaluation of stage, stages II and III were compared, which meant a comparison between operable and locally advanced tumors, since a majority of stage III patients had locally advanced tumors. Thus the patients with locally advanced cancers and stage III had by all expectations worse dis­ease-free survival. A statistically significant influence on the disease-free survival was also exerted by clinical and pathological lymph node status. Our univariate analysis has also shown that a worse prognosis was associated with negative estrogen receptors, while the presence of progesterone receptors in the tumor failed to provide prognostically relevant information. In our analysis menopausal status, pathological tumor size and grade of malignancy of invasive dueta! cancers did not show prognostic value for disease-free survival. The reason for the 
absence of prognostic value of pathological size and grade of tumors could be attributed to a relatively small number of patients included, as well as to a small number of events in the groups of tumors smaller than 2 cm and in moderately differentiated tumors. 
In multivariate models, both PAI-1 and 
PAI-2 showed an independent prognostic 
Radio/ 011col 1999; 33(1): 43-53. 

Boštnar Set al. / Urokinase plasminogen activator and its inhibitors 
value, the value of the former being some­what higher. In our study, only clinical tumor size and stage were stronger prognostic fac­tors than both inhibitors. In these two prog­nostic factors we actually used similarly formed groups, which are defined prevailing­ly by locally advanced disease, and thus pro­viding a similar information. In our study, clinical and pathological lymph node status have shown a lower prognostic power than both inhibitors, while estrogen receptors have lost their independent prognostic value to PAI-1 and PAI-2. Thus, our study has revealed that PAI-1 and PAI-2 are strong inde­pendent prognostic factors, and that only locally advanced disease provides a more rel­evant information on the outcome of disease. 
Independent prognostic value of PAI-1 for the disease-free survival of all breast cancer 
20 

patients was also established by German7 ,, Dutch6 , and French9 researchers. In all those studies, apart from pathological lymph node status, PAI-1 was found to be the strongest prognostic factor. Only the French study, which investigated not only PAI-1 but also the independent prognostic value of PAI-2, has confirmed that only pathological lymph node status has a stronger independent prog­nostic value.9 
Our study has therefore established the value of PAI-1 and PAI-2 contents in tumor cytosols for the prognosis of disease in breast cancer patients. Based on the results obtained, we believe that in the cases when only cytosol which does not enable a reliable uPA determination is available, PAI-1 and PAI-2 can provide a sufficient information for foretelling the outcome of disease. 
It is presumed that a combination of both inhibitors, and perhaps also their combina­tion with other components of the urokinase system or other proteinases might provide even better information for foretelling the outcome of disease, however, such an analy­sis would require a larger number of patients. It should be also necessary to establish the 
Radio! Onco/ 1999; 33(1): 43-53. 
prognostic value of urokinase system compo­nents in individual subgroups of patients, distributed according to menopausal status, lymph node involvement, hormonal status, etc. Such an approach would enable us to detect the patients at an increased risk of recurrence within the prognostically more favorable groups. Nevertheless, such an analysis as well would require a considerably larger number of patients. Apart from that, it would be interesting to find out whether immunohistochemically determined compo­nents of the urokinase system would provide a similar information, or would such determi­nation -likewise in the case of cathepsin D ­undermine the beliefs about their prognostic value.21 We plan to carry out a prospective study, which would touch upon at least some of the hypotheses presented here. 
References 
l. Dan0 K, Andreasen PA, Gr0ndahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. Plasrninogen activators, tissue degradation, and cancer. Adv Cancer Res 1985; 44: 139-266. 
2. 
Markus G. The relevance of plasrninogen activa­tors to neoplastic growth. Enzyme 1988; 40: 158-72. 

3. 
Schmitt M, Jiinicke F, Graeff H. Turnor-associated proteases. Fibrinolysis 1992; 6: 3-26. 

4. 
Borštnar S, Cufer T, Rudolf Z. The urokinase-type plasrninogen activator, its inhibitors and its recep­tor -the new prognostic factors in solid cancers. Radio! Oncol 1997; 31: 298-304. 

5. 
Duffy M, O'Grady P, Devaney D, O'Siorain L, Fen­nelly JJ, Lijnen HJ. Urokinase plasrninogen activa­tor, a rnarker for aggressive breast carcinornas. Cancer 1988; 62: 531-3. 

6. 
Foekens JA, Buessecker F, Peters HA et al. Plas­rninogen activator inhibitor-2: prognostic rele­vance in 1012 patients with prirnary breast cancer. Cancer Res 1995; 55: 1423-7. 

7. 
Gr0ndahl-Hansen J, Christensen IJ, Rosenquist C et al. High Levels of Urokinase-type plasrninogen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinornas are associated with poor prognosis. Cancer Res 1993; 53: 2513-21. 



Bošt11ar Set al. / Uroki11ase plas111i11ogen activator a11d its inhibitors 
8. 
Duffy M], Reilly D, O'Sullivan C, O'Higgins N, Fennelly JJ, Andreasen P. Urokinase plasminogen activator, a new and independent prognostic marker in breast cancer. Cancer Res 1990; 50: 6827-9. 

9. 
Duggan C, Maguire T, McDermott E, O'Higgins N, Fennelly ]J, Duffy MJ. Urokinase plasminogen activator and urokinase plasminogen activator receptor in breast cancer. lili J Ca11cer 1995; 61: 597-600. 

10. 
Grondahl-Hansen J, Peters HA, van Putten WLJ, Look MP, Pappot H et al. Prognostic significance of the receptor for urokinase plasminogen activa­tor in breast cancer. Cli11 Cancer Res 1995; 1: 1079­87. 

11. 
Foekens JA, Schmitt M, van Putten WLJ et al. Plasminogen activator inhibitor-1 and prognosis in primary breast cancer. J Ciin Oncol 1994; 12: 1648-58. 

12. 
Janicke F, Schmitt M, Pache L et al. Urokinase (uPA) and its inhibitor PAI-1 are strong and inde­pendent prognostic factors in node-negative breast cancer. Breast Cancer Res Treat 1993; 24: 195­208. 

13. 
Duffy MJ, Reilly D, McDermott E, O'Higgins N, Fennelly ]J, Andreasen PA. Urokinase plasmino­gen activator as a prognostic marker in different subgroups of patients with breast cancer. Cancer 1994; 74: 2276-80. 

14. 
Bouchet C, Spyratos F, Martin PM, Hacene K, 


Gentile A, Oglobine J. Prognostic value of uroki­nase-type plasminogen activator (uPA) and plas­minogen activator inhibitors PAl-1 and PAI-2 in breast carcinomas. Br] Cancer 1994; 69: 398-405. 

15. 
Kaplan EL, Meier P. Non-parametric estimation from incomplete observations. ] Am Stat Assoc 1958; 53: 457-81. 

16. 
Mante] N. Evaluation of survival data and two new rank order statistics arising in its considera­tion. Cancer Chemother Rep 1966; 50: 163-70. 

17. 
Cox DR. Regresion models and life-tables. J R Stat Soc Bull 1972; 34: 187-220. 

18. 
Jankun J, Merrick HW, Goldblatt PJ. Expression and localization of elements of the plasminogen activation system in benign breast disease and breast cancers. J Celi Biol 1993; 53: 135-44. 

19. 
R0nne E, H0yer-Hansen G, Briinner N et al. Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono-and polyclonal anti­bodies. Breast Cancer Res Treat 1995; 33: 199-207. 

20. 
Janicke F, Pache L, Schmitt M et al. Both the cytosols and detergent extracts of breast cancer tissues are suited to evaluate the prognostic impact of the urokinase-type plasminogen activa­tor and its inhibitor plasrninogen activator inhibitor type 1. Cancer Res 1994; 54: 2527-30. 

21. 
Duffy MJ. Proteases as prognostic rnarkers in can­cer. Ciin Cancer Res 1996; 2: 613-8. 


Radio/ 011col 1999; 33(1): 43-53. 

Rndiol Oncol 1999; 33(1): 55-8. 
Radiotherapy for choroidal neovascularisation of age-related 
macular degeneration 
Wolfgang Wagner1, Erik Beeke2, Peter Barsnick2 
1 Paracelsus Strahlenklinik, 2 Parace/sus-Klinik, Departnzent of Ophthalmology, Osnabriick, Germany 
Age-rclatcd macular dcgeneration caused by choroidal neovascularisation is an incrcasing proble111 in oph­thalmology. The rcsults oj the therapy in the past were poor or associated with a numbcr oj side ejjects. Rccently, some reports have slwwn a benejicial ejject oj low-dose irradiation. Therejore, we reviewed the data oj our patients included in a pilot study to conjirm the very preli111inary data in literature. Fourty­threc patients wcre irradiated with a linear accelerator (6 MV) at a total dose oj 16 Gy. Six months ajter irradiation, 69% oj our patients maintaincd or i111proved their visual acuity. We did not observc side ejjects ar any acute or late sequelae within a median jollow-up period oj 12 months. 
Key words: nzacular degeneration -radiotherapy, visual acuity 
Introduction 
Choroidal neovascularisation (CNV) is a major cause of severe loss of visual acuity in the patients with age-related macular degen­eration.1 Without therapy, the natura] course of this disease would result in the loss of patient's sight. 
In the past, laser photocoagulation was used as treatment method, but was, unfortu­nately, associated with further decrease in visual acuity.2 In the treatment of CNV, inter­feron was used systemically, but, so far, the 
Received 14 August 1998 
Accepted 7 December 1998 Correspondence to: Priv.-Doz. Dr. Wolfgang Wagner, Paracelsus-Strahlenklinik, Li.irmannstr. 38/40, 49076 Osnabrtick, Germany. Tel: + 49-541-966 4800; Fax: + 
substance has not proved to be effective, moreover, it may be that it has severe side effects.3 
Another approach is the surgical extirpa­tion although the treatment results are not too encouraging.4 Recently, some investiga­tors have reported of a beneficial effect of low-dose irradiation of the subretinal neovas­cular membranes in the CNV.5-13 The prelimi­nary results are encouraging and the number of side effects is small. 
Patients and methods 
Between September 1996 and July 1998, we treated 43 patients (25 women and 18 men) with radiotherapy for age-related macular 

49-541-681 137 degeneration. All patients gave their 
Wagner W e/ a/. / Irradiation in patienls with macular degeneration 
informed consent before irradiation. The median age at the tirne of treatment was 74 years (range 60-88 years). 
The right and the left eye were treated in 18 and 16 patients, respectively. In 9 patients, both eyes were involved. In all cases, the diag­nose was set on the basis of visual examina­tion (funduscopy) carried out by the ophthal­mologist. All patients were diagnosed as hav­ing the exudative variety of macular degener­ation. In 21 patients, angiography was used to determine the size of the neovascularisation. 
Irradiation was carried out using a linear accelerator of 6 MV photons. A total dose per patient was 16 Gy and was given in normal fractionation with single doses of 2 Gy 5 times a week. When both eyes were involved, opposed portals were used and the dose was specifically adjusted to the middle of the scull. In case of unilateral neovascularisation, a single irradiation field was used. In ali cases, we used a lens-sparing technique. 
The median follow-up tirne was 12 months. Each patient was clinically exam­ined before and immediately after the thera­py and then every 3 months. Ali patients included in this study were questioned about their subjective experience of radiotherapy, treatment results and side effects. 
Results 

After the completion of irradiation, visual acuity improved in 5 patients (11 %), in 35 patients (81 %), it was the same as before irra­diation and was worse in 3 patients. 
Six months after the irradiation, 30 patients (69%) maintained their visual acuity while in 9 patients (21 %), it was worse. Four patients were lost from the follow-up at the tirne of investigation. 
Objective changes of visual acuity could be observed only to a limited extent (median difference 0.005). In 5 patients, the moist macular degeneration changed to a dry one 
Radio/ Onco/ 1999; 33(1): 55-8. 
without affecting the acuity. In 2 patients, the bleeding occurred again 3 and 7 months after the completed irradiation. As to the side effects, we have not observed any acute or late sequelae after irradiation. 
In addition to the objective findings, the results of the interviews with the treated patients were the following results: 31 patients (72%) reported that irradiation had a beneficial influence on their acuity and 12 patients felt that irradiation did not improve their sight. 
Discussion 

Macular degeneration is an increasing prob­lem in ophthalmology. Today, about 5% of the population in their sixties are affected.1 The patients are handicapped by the loss of cen­tral vision and, therefore, their ability to read. 
Often, both eyes are affected to various degrees and at different times.14 Stage I shows a dry formation of senile membranes. A detachment of the pigment epithelium with retroretinar bleeding occurs within some weeks. In the exudative stage, disci­form lesions are observed. They consist of choroidal neovascularisation and spread into the retroretinal space as a thin layer between the retina and choroid. At this stage, patients often report seeing only shadows. Regression of the exudative changes causes complete atrophy of the central part of the retina. The therapy is stage-dependant.14 
In the early dry stage, no special therapy is recommended. The detachment of the pig­ment epithelium is treated with corticosteri­ods. Laser coagulation could be applied, but 
15-17 

causes scars and decreases the acuity. 2,Neovascular membranes cannot be treated with laser. In these cases, radiotherapy can be applied as alternative treatment. Irradia­tion seems to be able to stop further progress of the disease. 
Proliferating vascular cells have been 

Wagner W et al. / lrradiation in patients with 111acular degeneration 
known to be relatively sensitive to low doses of radiation. Irradiation may prevent the pro­liferation of endothelial cells of newly formed subretinal capillaries and induce obliteration of the aberrant new vessels.18 Some <lata that have appeared so far in the literature confirm the assumption that photon irradiation can be a beneficial tool in treating this disease. Chakravarthy et a/. treated macular degenera­tion with a total <lose of 10 or 15 Gy and recorded that the visual acuity was main­tained or improved in 78% of all patients.8 Hart and colleagues treated 41 patients with 10, 12 or 15 Gy and found no significant dif­ference in the effect of treatment of 3 differ­ent <lose regimes.10 Bergking et al. 5 evaluated the patients in a study on doses ranging between 8 and 24 Gy. The first group received 8 Gy in a single fraction. In this group, only 30% had stable visual acuity. In the second group, 50% of patients having received a <lose of 12 Gy had stable visual acuity after a follow-up of 18 months and in the third group having received a dose of 18 Gy, 40% of patients had stable visual acuity after the same follow-up period. In the group which had received 24 Gy, 80% of all patients 
had stable visual acuity. 
Hence, it is possible that there is a rela­tionship between total doses of irradiation and treatment results. But care should be taken not to exceed a total dose of 25 Gy in order to avoid an increase in side effects. 
On the other hand, some data published in literature report of the positive treatment results using proton beam irradiation. Yonemoto et a/. 13 reported that after a median follow-up of 11.6 months 58% of a total of 21 patients had an improved or stable visual acuity. So far, it has not been proved that, with regard to the treatment results, heavy particles such as protons are superior to nor­mal photon therapy. 
In our investigation, we recorded stable 
visual acuity objectively in 69% of all patients 

6 months after irradiation. After interviewing the patients, it was estimated that the effect of treatment was beneficial in 72% of cases. As no acute or late sequelae and no changes in the number of stable diseases were observed within the median follow-up time of 12 months, we believe that low-dose irradi­ation can be a good treatment policy of the patients with progressive CNV. Nevertheless, the definitive role of radiotherapy in patients with CNV is stil! to be defined by a control phase-III study. 
References 

l. Bressler NM, Bressler SB, Fine SL. Age-related macular degeneration. Surv Ophtha/1110/ 1988; 32: 375-412. 
2. 
Macular Photocoagulation Study Group. Laser photocoagulation of subfoveal neovascular lesions in age-related macular degeneration. Results of a rendomized clinical tria!. Are/z Ophtha/1110/ 1991; 9: 1220-31. 

3. 
Thoelen A, Menozzi M, Huber C, Messmer E. Treatment of choroidal neovascularization in age­related macular degeneration with interferon alpha-2a: a short term, nonrandomized pilot study. Ger J Ophtha/111011995; 3: 137-43. 

4. 
Thomas MA, Dickinson JD, Melberg NS, lbanez HE, Dhaliwal RS. Visual results after surgical removal of subfoveal choroidal neovascular mem­branes. Ophtha/1110/ogy 1994; 8: 1384-96. 

5. 
Bergink GJ, Deutman AF, von den Broek JE, van Daal WA, van der Maazen RM. Radiation therapy for age-related subfoveal choroidal neovascular membranes. A pilot study. Doc Ophthalmol 1995; 90: 67-74. 

6. 
Bergink GJ, Deutman AF, van den Broek JF, van Daal WA, van der Maazen RW. Radiation therapy for subfoveal choroidal neovascular membranes in age-related macular degeneration. A pilot study. Graefes Arch Ciin Exp Ophtlialmo/ 1994; 10: 591-8. 

7. 
Bergink GJ, Hoyng CB, van der Maazen RW, Deut­man AF, van Daal WA. Visual acuity and scar size in eyes with age-related subfoveal choroidal neo­vascular lesions, 30 months after radiation thera­py. Doc Ophtlialmol 1996; 1: 61-75. 

8. 
Chakravarthy U, Houston RF, Archer DB. Treat­ment of age-related subfoveal neovascular mem­branes by teletherapy: a pilot study. Br J Oplzthal­111011993; 5: 265-73. 



Radio/ 011co/ 1999; 33(1): 55-8. 

Wagner W et al. / Irradiation in patie11ts with macular degeneration 
9. 
Harino S, Oshima Y, Tsujikawa K, Oh A, Sugimo­to K, Murayama S, et al. Treatment of age-related subfoveal choroidal neovascularization by low­dose external radiation: a preliminary study. Nip­pon Ganka Gakkai Zasshi 1997; 4: 341-8. 

10. 
Hart PM, Chakravarthy U, Mac Kenzie G, Archer DB, Houston RF. Teletherapy for subfoveal choroidal neovascularisation of age-related macu­lar degeneration: results of follow-up in a non-ran­domized study. Br J Opht/,a/111011996; 12: 1046-50. 

11. 
Matsuhashi H, Takahashi D, Noda Y, Mariya Y, Tarusawa N, Yoshimoto H, et al. Low-dose radia­tion, therapy for choroidal neovascularization in age-related macular degeneration. Nippon Ganka Gakkai Zasshi 1996; 10: 803-9. 

12. 
Sasai K, Murata R, Mandai M, Takahashi M, Ogura Y, Ngata Y, et al. Radiation therapy for ocu­lar choroidal neovascularization (phase !/II study): preliminary report. Int J Radia/ Oncol Biol Phys 1997; 1: 173-8. 

13. 
Yonemoto LT, Slater JD, Friedrichsen EJ, Loredo LM, Ing J, Archambeau JO, et al. Phase !/II study of proton beam irradiation for the treatment of subfoveal choroidal neovascularization in age­related macular degeneration: treatment tech­niques and preliminary results. Int J Radia/ Oncol Biol Phys 1996; 4: 867-71. 


14. 
Schleicher UM, Andreopoulos D, Wolf S, Schmachtenberg A, Ammon J, Reim M. Radio­therapy of macular degeneration. Experience with two fractionation schemes. Front Radiat Ther Oncol 1997; 30: 253-8. 

15. 
Coscas G, Soubrane G, Ramahefasolo C, Fardeu 


C. Perifoveal laser treatment for subfoveal choroidal new vessels in age-related macular degeneration. Results of a randomized clinical tria!. Arch Ophtha/111011991; 9: 1258-65. 
16. 
Maculai· Photocoagulation Study Group. Laser photocoagulation of subfoveal recurrent neovas­cular lesions in age-related macular degeneration. Results of a randomized clinical tria!. Arch Oph­tha/1110/ 1991; 9: 1232-41. 

17. 
Macular Photocoagulation Study Group. Visual outcome after laser photocoagulation for sub­foveal choroidal neovascularization secondary to age-related macular degeneration. The influence of initial lesions size and initial visual acuity. Arch Ophthalmol 1994; 4: 480-8 

18. 
Archer DB, Amoaku WMK, Gardiner TA. Radia­tion retinopathy -clinical, histopathological, ultra­structural and experimental correlations. Eye 1991; 5: 239-51. 


Radio/ Oncol 1999; 33(1): 55-8. 

Radio/ Oncol 1999; 33(1): 59-61. 
Second malignancy after radiotherapy for seminoma 
Pascal R Fuchshuber, R Jeffrey Lee, 1 Brian McGrath, John F Gibbs, William G Kraybill, Gary M Proulx1 
1 Division oj Surgical Oncology and Radiation Medicine, Roswell Park Cancer Institute, Buffalo, New York, USA 
A patient wlw developed a malignant peripheral nerve sheath tumor in the field oj radiation 19 years after radiotherapy far stage I seminoma is presented. Data from recent population-based studies evaluating the risk oj second malignancies in this group oj patients is discussed. This case report illustrates the need far judicious evaluation oj adjuvant radiotherapy in early stage seminoma patients. 
Key words: seminoma-radiotherapy; radiotherapy-adverse effect; neoplasms, second primary; nerve 
sheath tumor; soft tissue sarcoma 
Introduction 

The role of adjuvant radiotherapy for stage I seminoma is controversial. Although the relapse free survival is greater than 95% with adjuvant therapy, there is no benefit in over­all survival since patients who relapse are sal­vaged with treatment. Additional issues include the frequency of follow-up studies, maintenance of fertility, and the risk of radia­tion induced second malignancies. We report a patient who developed a malignant sarco­ma in the radiation field 19 years following adjuvant radiotherapy for seminoma. 
Received 3 July 1998 
Accepted 10 October 1998 Correspondence to: R. Jeffrey Lee, MD, Division of Radiation Medicine, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263. Phone: +1 716 845 3172; Fax: +1 716 845 7616. 
Case report 

A 45-year old man presented 19 years after left orchiectomy and radiotherapy for a stage I seminoma with severe pelvic pain and a dense left sciatic nerve palsy. Computer tomography (CT) showed a 8.5 cm enhancing heterogeneous mass extending to the superi­or aspect of the sacrum (Figure 1). An ultra­sound-guided biopsy revealed a high grade malignant peripheral nerve sheath tumor. Staging chest and abdominal CT showed no additional lesions. The patient received 3000 cGy of external beam radiation. The original simulation films show the radiation fields (Figure 2), which includes the paraaortic, left iliac and pelvic region. Given this previous radiation treatment, it was now felt that neoadjuvant radiotherapy was not indicated. A curative surgical resection was attempted which included a modified left interna! 
Fuchshuber PR et al. / Second malignancy after radiotherapy for seminoma 

Figure l. CT of the pelvis demonstrating a left pelvic mass. Tissue biopsy revealed a malignant peripheral nerve sheath tumor. 

hemipelvectomy with sacrifice of the sciatic nerve. Intraoperative margin assessment was negative for tumor, however, final pathology revealed a positive microscopic margin. The patient refused adjuvant radiation secondary to the risks of bladder and bowel toxicity. Ten weeks postoperatively the patient developed lung metastases. Treatment with Adriamycin was initiated. 
Discussion 
Iatrogenic carcinogenesis due to ionizing radiation is a well established observation. The probability of secondary malignancies increases with the dose. A threshold is not known, however, and doses as low as 1000 cGy have been associated with secondary malignancies .1 
While radiation therapy is an integral part of the primary treatment for many cancers, 

Fuchshuber PR et al. / Second malignancy after radiothcrapy far seminoma 
its role in stage I testicular seminoma remains controversial. Surveillance after orchiectomy may be a safe alternative to adju­vant radiotherapy, if one is prepared to accept a 15 to 20% recurrence rate. If all patients receive adjuvant radiotherapy, recur­rences can be reduced to 2 to 4% which can be treated with systemic chemotherapy.2 If one elects surveillance, 85% of patients are cured. The remaining 15% of patient will relapse and undergo retroperitoneal irradia­tion only (10%), chemotherapy only (3%), or combined chemoradiation (2%).2 Therefore, with surveillance alone 1 to 2% more patients will require chemotherapy but the majority of patients will be spared radiotherapy. Young patients wishing to remain fertile may elect surveillance, whereas patients anxious about the higher relapse rate may opt for adjuvant radiotherapy. 
The issue of radiation-induced malignan­cies in seminoma patients remains controver­sial.3 While cases such as the one presented here support the entity of postirradiation malignancies in seminoma patients, popula­tion based studies have resulted in conflict­ing reports on the risk for cancer following 
5 


adjuvant radiotherapy.4, Whereas studies have found no evidence of an increased risk for second malignancies after adjuvant radio­therapy for seminoma, others have estimated the risk for postirradiation sarcoma in this group of patients at 0.003% to 0.8%.1 The largest population based study on this sub­ject has recently been published by the National Cancer Institute. It has documented a significantly elevated risk of second malig­nancies in seminoma patients treated with radiotherapy.6 The cumulative risk of second malignancies at 25 years was 18.2 % for men with seminoma compared with 9.3 % in the general population.6 The expected occur­rence of connective tissue tumors in this group of patients was approximately 4 times higher in seminoma patients 20 years after initial treatment. The risk remained elevated throughout the follow-up period.6 The medi­an latency to tumor development was approximately 10 to 15 years. 
Differences in treatment modalities, stage distribution, and surveillance for early stage seminoma patients weaken the conclusions of most studies examining radiation carcino­genesis. Unfortunately, the prognosis for patients who develop postirradiation sarco­mas is poor, particularly for those located in the pelvis, with a 5 year survival rate of less than 10%.1 
In conclusion, this case illustrates the need for the careful evaluation of risks and benefits of adjuvant radiotherapy in early stage seminoma patients. 
References 

1. 
Mark RJ, Poen J, Iran LM, Yao SF, Selch MI, Park­er RG. Postirradiation sarcoma. A single-institu­tion study and review of the literature. Cancer 1994; 73: 2653-62. 

2. 
Thomas GM and Williams SD. Principles and prac­ticc oj radia/ion oncology. 3rd Edition. Philadelphia: Lippincott-Raven Publishers; 1997. p. 1695-715. 

3. 
Warde PR, Gospodarowicz MK, Panzarella T. Stage I testicular seminoma: Results of adjuvant irradia­tion and surveillance. J Cli11 Oncol 1995; 13: 2255­62. 

4. 
Chao C, Lai P, Michalski J, Perez C. Second malig­nancy among seminoma patients treated with adjuvant radiation therapy. In/ J Radia/ Oncol Biol Plzys 199; 33: 831-5. 

5. 
Van Leeuven FE, Stiggelbout AM, Van den Belt­Dusebout AW, Noyon R, Eliel MR, van Kerkhoff EA, et al. Second cancer risk following testicular cancer: A follow-up study of 1.909 patients. J Ciin Oncol 1993; 11: 415-24. 

6. 
Travis LB, Rochelle E, Storm H, Hall P, Holowaty E, Van Leeuwen FE, et al. Risk of second malig­nant neoplasms among long-term survivors of tes­ticular cancer. JNCI 1997: 89: 1429-39. 


Radio/ O11co/ 1999; 33(1): 59-61. 

Radio/ Oncol 1999; 33(1): 63-8. 
Sarcomas of the bladder: A case report with a review of the literature 
Gary M Proulx1 , John F Gibbs2, R JeffreLee1 , 
r Satish Velagapudi3 , Robert Huben 

1Department oj Radiation Medicine, 2Department oj Surgical Oncology, Sarcoma Service, 3Departrnent oj Urologic Oncology, Roswe/l Park Cancer Institute, Buffalo, USA 
Sarcomas oj the bladder are rare tumors wit/1 decisions about treatment and understanding oj outcome based on limited reports from single institutions. Consequently, neither a standard staging system nor stan­dard treatment exists far this disease. A retrospective review of the medica/ records at our institution from January 1960 to December 1997 far sarcomas of the bladder and a review of the literature were made to better understand the natura/ history and treatment outcome of these tumors. We report on one case treated with curative intent and summarize the experience of other institutions. If disease is localized to the bladder, then surgery a/one, either by tata/ ar partia/ cystectomy ar, alternative­ly, a multimodality approach using a combination of treatment with partial cystectomy and local radiation with chemotherapy to address risk of systemic disease can offer patients a good chance far cure 
Key words: bladder neoplasms -drug therapy -radiotherapy -surgery; sarcoma 
Introduction 

Sarcomas are rare with an approximate inci­dence of only 7000 cases per year in the Unit­ed States. Those involving the genitourinary tract, not including rhabdomyosarcomas, account for less than 5% of soft tissue sarco­mas and only 1-2% of all genitourinary tumors. Bladder sarcomas, in particular, tend 
Received 27 July 1998 
Accepted 10 September 1998 Correspondence to: Gary M. Proulx, M.D., Depart­ment of Radiation Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. 
to occur at the extremes of life with a bimodal distribution of less than age sixteen and greater than age sixty.1 Most patients present with painless gross hematuria with other common symptoms being urinary fre­
3 5 

quency, hesitancy and/or retention.2, -In adults , severa! histologic subtypes have been reported. The most frequent histology in adults appears to be leiomyosarcoma with other less frequent ones including malignant fibrous histiocytoma, angiosarcoma, fibrosar­coma, extraosseous osteogenic sarcoma and 
6 10 

sarcoma NOS.3 ,-Rhabdomyosarcomas (RMS) are the most common pediatric blad­
Proulx GM et al. / Adult bladder sarcomas 
der sarcoma being clinically dissimilar from other soft tissue sarcomas and are generally managed according to national protocols. Although RMS occurs mainly in the pediatric population, forty cases were reported in a very early paper on bladder sarcomas in adults.1 
No clear etiology exists for sarcomas of the bladder. Unlike bladder carcinomas which have severa! established risk factors (e.g. tobacco), sarcomas of the bladder have not been shown to have causal relationships to such factors. Very early reports have suggest­ed that embryologic mesenchymal remnants are perhaps the origin of bladder sarcomas.11 More recent literature has suggested a genet­ic link to sarcomas, including those of the 
14 
genitourinary tract.12-Also sarcomas aris­ing as second malignancies have been report­ed from prior radiation treatment and chemotherapy treatment with the alkylating agent, cyclophosphamide.15-18 
Its rarity prevents accruing enough patients to accurately assess treatment effica­cies and to analyze characteristics of patients for prognostic indicators. Consequently, rec­ommendations about treatment have been conflicting and based on small numbers from institutional experiences. We report a case diagnosed and treated at Roswell Park to add to the relatively few cases that have been reported and have reviewed the literature to assist in treatment decisions. 
Case report 
An 80 year old white male developed urinary frequency, urgency and painless gross hema­turia. He underwent an intravenous pyelo­gram and cystoscopy. These studies revealed a mass within the bladder which was removed by partial cystectomy. The mass measured five centimeters in greatest dimension and the pathology showed it to be a leiomyosarco­ma of intermediate grade. The remainder of his workup was negative for metastatic dis­ease which included laboratory and radi­ographic studies. He received postoperative treatment using high energy photons to irra­diate his whole pelvis to 4800 cGy to cover the bladder and its draining lymphatics and then boost treatment to the bladder only for an additional 800 cGy for a total dose of 5600 cGy. The radiation was given using 400 cGy per treatment with treatments given twice daily using an anterior-posterior field to the whole pelvis. This same treatment was used for the boost with the volume reduced to cover the bladder plus a margin. The patient remained disease free at one year follow up. 
Discussion 
Bladder sarcomas are rare tumors which makes treatment decisions and assessing treatment outcome difficult. Having a staging system that better characterizes bladder sar­comas would help with both treatment deci­sions and evaluating outcome. Currently, such a staging system is lacking. These tumors are presently staged according to the same systems utilized for bladder carcino­mas, namely the Marshall-Jewet or American Joint Committee on Cancer (AJCC) TNM transitional cell bladder carcinoma systems. These staging systems are very effective with non-sarcomas of the bladder because they relate the close relationship between muscle invasion and risk of regional node and/or dis­tant disease.19 Bladder sarcomas, however, originate within the submucosal mesenchy­mal layer which would automatically stage them as invasive and place them at high risk for nodal and/or distant disease with a result­ing poor prognosis. With the exception of RMS, this is not the case with sarcomas of the bladder which have a relatively low risk of regional nodal involvement. Using the AJCC soft tissue sarcoma staging system, on the other hand, would perhaps offer a better 
Radio/ 011co/ 1999; 33(1): 63-8. 

Prou/x GM et ni./ Adult bladder sarco111as 
approach since its predominate prognostic factor is grade. 
However, tumor size would not adequate­ly address bladder sarcoma. Swartz et al. have shown that the amount of bladder involvement is a prognostic indicator. We propose in this paper a staging system that 
Table l. Proposed staging system for sarcomas of thc bladder 
Histologic subtype Leiomyosarcoma Rhabdomyosarcoma Other 
Grade: low, intermediate, high 
Size Bulky (>30% bladder involvement) Nonbulky Pelvic sidewall involvement Adjacent organ involvement 
Regional nodal involvement 
Distant metastasis 

incorporates these apparent associated prog­nostic factors (Table 1) to allow better follow­up of patients and establish a multidiscipli­nary approach to bladder sarcomas. Specifi­cally, mitotic activity, a major determinant of grade, has been shown to be an independent prognostic indicator and correlate well with developing metastases and overall sur­vival. 20, 21 Others include the extent of locoregional involvement and histologic sub­grouping. 
With respect to treatment and outcome of sarcomas of the bladder, surgery has remained the mainstay and improvements in outcome have been reported in the modern literature with survival as high as 63% at 5 years.22 Sen et a/. evaluated 13 patients at the Mayo Clinic between 1970 and 1985 with radical cystectomy with or without preopera­tive radiation.2 3 They had five patients with leiomyosarcoma, three with rhabdomyosar­coma, and five with carcinosarcoma. 
Two of the 5 patients with leiomyosarcoma were treated with preoperative irradiation. One (pathologic stage Bl) of these two patients was treated with radical cystectomy and preoperative irradiation of 5000 cGy and died at sixteen months after developing local and distant disease. The other (PS Bl) was treated preoperatively with 6000 cGy and is free of disease seventy-eight months after completion of treatment. Two of the five were treated with radiation alone with doses of 6500 cGy and 6000 cGy and died at eighteen months and forty-six months respectively after developing both local and distant dis­ease. No clinical stage was given for either but neither was felt to be surgical candidates. One patient (PS A) had a radical cystectomy alone and is free of disease at eighty-six months. The authors conclude from their experience that those patients felt to be cura­tive should be treated with radical cystecto­my with or without preoperative irradiation regardless of histology and that with modern techniques for staging and treatment, patients have a much better prognosis today than patients described in the older litera­ture. 
Another series by Swartz et al. on leiomyosarcoma of the bladder reported on ten cases. Pathologic staging was not indicat­ed in the paper so that no correlation of pathologic stage and treatment outcome can be made. One patient treated with partial cystectomy and postoperative radiotherapy (<lose not given) for inadequate margins has remained alive without disease at 5 years of follow-up.12 Three others treated with partial cystectomy alone with adequate margins are alive without disease at 6, 6 and 9 years. Only one patient was treated with definitive radio­therapy after an initial misdiagnosis of squa­mous celi carcinoma and recurred locally after 4 years. The other patients in the series had either radical cystectomy (n=3), partial 
Radio/ 011co/ 1999; 33(1): 63-8. 

Proulx GM et ni. / Adull bladder sarcomas 
=
cystectomy with chemotherapy (nl), ileal 
conduit (n=l), or biopsy alone (n=l), and ali are dead of either postoperative complica­tions or disease progression. The authors in this paper felt that if adequate margins can be obtained, then partial cystectomy is the treatment of choice, and that radical cystecto­my should be performed only for more exten­sive lesions where adequate margins are not obtainable. 
They did not feel that enough information exists on the role of either postoperative radi­ation or adjuvant chemotherapy with this dis­ease to justify their use as part of initial cura­tive treatment but rather could be utilized in the setting of recurrent and/or metastatic dis­ease. 
Eleven patients with leiomyosarcoma were reported by Ahleringer et az. 2, 7 of whom had bladder as the primary site and 4 with prostate. Patients with nonbulky disease of the bladder were treated with surgical resec­tion and given adjuvant chemotherapy and external beam irradiation if the margin or nodes were positive. Bulky disease, defined as greater than 30% of bladder involvement, received preoperative chemotherapy with or without postoperative irradiation. None of their patients had pathologically positive nodal disease. Six of the 7 bladder patients are alive without evidence of disease (range of 35-97 months) with 1 lost to follow-up. The authors concur with Swartz et al. 12 in suggest­ing that partial or subtotal cystectomy for smaller tumors to avoid urinary diversion is justified as long as negative margins are achieved, and that, based on the 3 patients treated for stage C prostate involvement, radiotherapy with doses of between 4500 cGy and 5000 cGy be used for microscopic resid­ual disease after removal of bulky disease. They also further suggest adopting preopera­tive chemotherapy for cytoreduction using agents such as cisplatin and doxorubicin for bulky or transmural disease and postopera­tive chemotherapy for either responsive tumors to preoperative chemotherapy or pathologic 3A patients. 
A series from Memorial Sloan Kettering reviewing their experience with urologic sar­comas between 1982 and 1989 found 6 cases of leiomyosarcomas of the bladder.24 The authors discuss the local and distant recur­rences as well as survival <lata according to histologic grade, size and margin status after surgery, but they make no distinction between the different subtypes of bladder sarcoma. 
Thus no conclusions as to differences in outcome between leiomyosarcomas and other histologies can be made. No patient received irradiation as part of their treatment but two had treatment with surgery and chemothera­py. The 3 rhabdomyosarcomas were treated on a rhabdomyosarcoma protocol with multi­modalities of surgery, chemotherapy and radiation. Two patients with small (< 2 cm) leiomyosarcomas, 1 of which was a low grade and the other a high grade tumor, underwent complete transurethral resection alone with local control reported with 7 years follow-up. As a group, when compared with other uro­logic sites, sarcomas of the bladder had the highest 5-year survival at 80%. The authors show that urologic sarcomas in general share similar prognostic indicators as with sarco­mas at other sites; namely, with increasing grade, size and depth, both local recurrences and metastases increase. 
Our review of the literature shows that varying modes of managing these rare tumor exists. This is not surprising since personal experiences and extrapolation of treatments tend to control management decisions when there is little <lata available to assist in the decision making process. The location of the tumor would seem to occur anywhere within the bladder by the published reports, although the trigone region appears to have 
26 29 
the highest predilection. 25, -This may 
account for the varying results that are achieved with using a partial resection alone. 
Radio! Oncol 1999; 33(1): 63-8. 

Proulx GM et al. / Adu/1 /Jladder snrcomas 


Despite the differences that exist in the out­come between series, most series seem to suggest that a subtotal cystectomy as opposed to a radical cystectomy can achieve good results as long as adequate margins are achievable. More aggressive surgery appears to be indicated for large, bulky tumors. How­ever, the addition of radiation for positive margins after partial cystectomy and/or chemotherapy also appears to offer good results and thus avoid the need for removing the bladder.2 The results of Russo et al. and Suit et a/. would suggest that, like sarcomas at other sites, a multimodality approach for uro­logic sarcomas may provide better functional outcome and durable local contro!.24, 30 The case we present in this paper, although with short follow-up, is a clear example of how properly selected patients can undergo con­servative management with this disease involving the bladder, as has been shown with transitional celi tumors of the bladder, having disease controlled without the need for removing the bladder.31 Adopting a stan­dard staging system would allow for optimiz­ing treatment in a multidisciplinary fashion by basing treatment decisions on prognostic factors and aid in the design of future treat­ment protocols. 
References 
l. Narayana AS, Loening S, Weimar GW, Culp DA. Sarcoma of the bladder and prostate. J Liro/ 1978; 119: 72-6. 
2. 
Ahlering TE, Weintraub P and Skinner DG. Man­agement of adult sarcomas of the bladder and prostate. J Uro/ 1988; 140: 1397-9. 

3. 
Mackenzie AR, Whitmore Jr. WF, Melamed MR. Myosarcomas of the bladder and prostate. Cancer 1968; 22: 833-44. 

4. 
Mills SE, Bova GS, Wick MR, Young RH. Leiomyosarcoma of the urinary bladder: A clinico­pathologic and immunohistochernical study of 15 cases. Am J Surg Pntil 1989; 13: 480-9. 

5. 
Silbar JO, Silbar SJ. Leiomyosarcoma of bladder. 


Three case reports and a review of literature. J Liro/ 1955; 73: 103-11. 

6. 
Aragona F, Ostardo E, Prayer-Galetti T, Piazza R, Capitanio G. Angiosarcoma of the bladder: A case report with regard to histologic and immunohisto­chemical findings. Eur Uro/ 1991; 20: 161-3. 

7. 
Bcrenson RJ, Flynn S, Freiha FS, Kempson RL, Torti FM. Primary osteogenic sarcoma of the blad­der. Case report and review of the literature. Cnn­cer 1986; 57: 350-5. 

8. 
Oesterling JE, Epstein JI, Brendler CB. Myxoid malignant fibrous histiocytoma of the bladder. Ca11cer 1990; 66: 1836-42. 

9. 
Phan CN, Wilkinson M, Cohen MB, Dunn S, Car­roli P. Primary osteogenic sarcorna of the urinary bladder successfully treated with combination therapy. Urology 1994; 44: 771-4. 

10. 
Tungekar MF, AI Adnani MS. Sarcomas of the bladder and prostate. The role of irnmunohisto­chemistry and ultrastructure in diagnosis. Europ Llrol 1986; 12: 180-3. 



l'l. Cohen JS. Turnors of muscle type. Report of a group of cases, with special reference to rnetasta­sis of leiomyosarcoma to the brain. Arcil Pnth 1932; 13: 857-67. 
12. 
Swartz DA, Johnson DE, Ayala AG and Watkins, DL. Bladder leiomyosarcoma: A review of 10 cases with 5 year follow-up. J Llrol 1985; 133: 200. 

13. 
McKeen EA, Bodurtha J, Meadows AT, Douglass EC, Mulvihill JJ. Rhabdomyosarcorna complicating multiple neurofibrornatosis. J Ped 1978; 93: 992-3. 

14. 
Varley JM, Evans DGR, Birch JM. Li-Fraumeni syn­drorne 



a molecular and clinical review. Br J Can 1997; 76: 1-14. 
15. 
Brady MS, Gaynor JJ, Brennan, MF. Radiation­associated sarcorna of bone and soft tissue. Arc/1 Surg 1992; 127: 1379-85. 

16. 
Jacobsen GK, Mellemgard A, Wngelholm, SA, Moller H. Increase incidence of second primary cancer following testicular cancer. Eur J Can 1993; 29: 664-8. 

17. 
Robinson E, Neugit AI, Wylie P. Clinical aspects of postirradiation sarcomas. JNCI 1988; 80: 233­40. 

18. 
Travis LB, Rochelle E, Storm H, Hall P, Holowaty E, et al. Risk of second malignant neoplasms among long-term survivors of testicular cancer. JNCI 1997; 89: 1429-39. 

19. 
Messing EM, Catalona W. Urothelial tumors of the urinary tract. In: Walch PC, Retick AB, Vaughan 


Radio/ Onco/ 1999; 33(1): 63-8. 

Proulx GM et a/. / Adult bladder sarcomas 
ED Jr., Wein AJm, editors. Cambel/'s Urology. 7th Ed. Philadelphia: WB Saunders; 1998. p. 2327-410. 

20. 
Levine EG, Brooks JSJ, Spellrnan J, Velagapudi S. Genitourinary sarcomas. In: Raghavan D, Schner HI, Leibel SA, Lange PH, editors. Principles and practice oj genitourinary oncology. Philadelphia: Lip­pincott-Raven; 1997. p. 1007-20. 

21. 
Mills SE, Bova GS, Wick MR, Young RH. Leiomyosarcorna of the urinary bladder: A clinico­pathologic and imrnunohistochernical study of 15 cases. Am J Surg Path 1989; 13: 480-9. 

22. 
Tsudarnoto T and Lieber MM. Sarcomas of the kidney, urinary bladder, prostate, sperrnatic cord, paratestis and testis in adults. In: Raaf JH, editor. Management of soft tissue sarcornas. Chicago: Year Book Medica! Publishers; 1989. 

23. 
Sen SE, Malek RS, Farrow GM and Lieber MM. Sarcorna and carcinosarcorna of the bladder in adults. J Ural 1985; 133: 29. 

24. 
Russo P, Brady MS, Conlon K, Bajda SI, Fair WR, Herr HW, Brennan MF. Adult urological sarcoma. J Llrol 1992; 147: 1032-7. 

25. 
Young RH. Pathology oj the urinary bladda New York, Edinburgh, London: Churchill Livingstone; 1989. 


26. 
Weitzner S. Leiornyosarcorna of urinary bladder in children. Urology 1978; 12: 450. 

27. 
Bohn AW, Urwiller RD and Pantos TG. Leiornyosarcorna of the urinary bladder with review of the literature. Henry Ford Hosp. Med Bul/ 1962; 10: 445. 

28. 
Wilson TM, Fauver HE and Weigel JW: Leiornyosarcoma of urinary bladder. Urology 1997 ; 13: 565. 

29. 
Pathology of malignant turnors In: The biology and c/inica/ management oj bladder cancer. Oxford, Lon­don, Edinburgh, Melbourne: Blackwell Scientific Publications; 1975. p. 104-8. 

30. 
Suit HD, Rosenberg AE, Harmon DC, Mankin HJ, Wood WC and Rosenthal D. Soft tissue sarcornas. In: K. Halnan and K. Sikora, editors. Treatment oj cancer. 2nd Ed. London: Chapman and Hall Ltd.;1990. p. 657-77. 

31. 
Kaufman DS, Shipley WU, Griffin PP, Heney NM, Althausen AF, and Efird JT. Selective bladder preservation by cornbination treatrnent of invasive bladder cancer. NEJM 1993; 329: 1377-82. 


Radio/ Oncol 1999; 33(1): 63-8. 
Rndiol Oncol 1999; 33(1): 69-75. 
Epithermal neutron beam for BNCT at the JSI TRIGA reactor -modelling and experimental verification 
Marko Maucec, Bogdan Glumac, Jože Rant, Edvard Krištof 
Jožef Stefan Institute, Reactor Physics Division, Ljubljana, Slovenia 
It has been reported that satisfactory thermal/ epithennal neutron beams far Eoron Neutron {:apture Ihera­py (BNCT) could be designed at TRIGA research reactors, which are generally perceived as being saje to install and operate in populated areas. This contribution presents the most recent research activities in this field at the Jožef Stefan Institute TRIGA reacto1; where an epithermal neutron beam far BNCT is being developed. Experimental verification oj Mante Carla simulation results proves the quality and wide applic­ability oj the developed 3-D model, particularly oj the reactor core and irradiation channels. Due to high attenuation oj the epithemzal neutron flux ( cJ>i = 4.lx106 n/ cm2s, two orders oj magnitude below the ther­
ep 

apeutic limit) the irradiation facility in the current stage oj development is not appropriate far the c/inica/ BNCT treatments. Furthermore, the contribution oj the 2.5 mm air gap surrounding the facility is unaccept­ably high, thus making the relative gamma dose (Dy/cJ>e) almost 60-times higher than therapeutically rec­
p
ommended. Nevertheless, using gamma shielding oj Pb ar Bi and LiF ar Li2CO3 (thennal neutron cut-off), the quality oj the epithermal neutron beam would be significantly upgraded and become appropriate far in vitro studies oj baron compound transport in malignant tumour cel/s ar smaller laboratory animals. 
Key words: baron neutron capture therapy; neoplasms-radiotherapy; nuclear reactors 
Introduction 

];ioron Neutron .C.apture Iherapy (BNCT)1 is a bimodal treatment that offers the potential of a highly selective radiation effect -by a parti­cles -while sparing normal tissues. Brain tumours, particularly glioblastoma multi­forme (GBM), were chosen as the initial tar-
Received 5 October 1998 Accepted 10 December 1998 
Correspondence to: Marko Maucec, MSc, Jožef Stefan Institute, Reactor Physics Division, Jamova 39, POB 3000, 1001 Ljubljana, Slovenia. E-mail: marko .maucec@ijs.si 
get far BNCT. GBM is an extremely lethal cancer, with no significant advances in thera­py in the last two decades. Almost all patients die within two years, even with the best efforts using surgery, external beam therapy and chemotherapy.1, 2 
The central feature in effective BNCT is the selective delivery, concentration and build-up of the naturally occurring lOB iso­tope in tumour tissue, using one or more advanced drug delivery systems (DDS) such as monoclonal antibody carriers or liposomal deliveries.3-5 As the tumour is irradiated with low energy neutrons (epithermal neutrons 
Maucec M ct a/. / Epithcrmal ncutron bcam far BNCT at ]SI TRI GA rcactor 
with 0.4 eV<E<l0 keV become thermalized in the surrounding healthy tissue), there is a higher likelihood of the 10B nucleus absorb­ing them than the nuclei of any other ele­ments normally present in tissues. The baron nucleus become unstable and immediately splits into two recoiling ionizing particles, an particle (i.e., a helium nucleus) and a lithium nucleus: 
n + 10B . 11B* . 4He + 7Li + y 
These products of the BNCT reaction ­10B(n,a)7 Li -are very damaging to tissue, but of short range (the pathlength of particles is about one celi diameter, which is around 10 µm) and are confined to the immediate vicin­ity of the boron-containing compound which, hopefully, should be concentrated in the tumour. A major appeal of BNCT is that 7Li and energetic a particles are produced by a fission reaction following neutron capture. These heavy particles carrying 2.79 MeV of energy, have a very high ionization density. Another advantage is that they can affect dividing and nondividing tumour cells alike -tumours are known to have a large number of viable but nonactive cells. 
Up to the present NCT research in Slove­nia has been carried out in a provisional irra­diation system, consisting of a 11 dry celi" adja­cent to the thermalizing column (Figure 1) at Jo_ef Stefan Institute (JSI) 250 kW TRIGA Mark II research reactor. The 11 dry celi" (origi­nally a pool-type storage facility) is a unique advantage of the JSI TRIGA reactor, with approx. ground plan dimensions of 3x3 m2 and additional neutron protection, currently refurbished for experimental purposes. The radiative field cross-section of the thermaliz­ing column is approx. 60x60 cm2 , and is rather homogene due to the substantial dis­tance from the core fuel elements. The irradi­ation field of the experimental set-up is approx. lOxlO cm2 , thus enabling irradiation of 9 foils with specimens at the tirne. The experimental set-up is mainly used for in vitro studies in a mixed neutron/gamma irra­diation field, with the aim of determining the radiosensitivity of two cell lines: mouse B16F1 melanoma and human MCF7 breast 
7 
carcinoma. 6, 
Unfortunately, the contribution of fast neutrons (18.4%) as well as y-rays (23%) to the total effective dose in the current experimen­tal system is substantial.6 Since this has a strong negative influence on the verification of 10B or thermal neutron effects, as well as on the mortality of prepared cell cultures, the development of an irradiation facility with optimized beam properties (minimised fast neutron and gamma dose and maximised epithermal neutron flux) is indispensable. Hence Mante Carla modeling, development as well as experimental verification of the epithermal neutron beam in the radia! chan­nel of the JSI TRIGA reactor is presented in this paper. 
Methods 
Mante Carla modeling oj the BNCT irradiation jacility in the radia/ channel oj the TRIGA reac­tor 
The general purpose MCNP4B Mante Carla code was used for modeling of the TRIGA reactor, together with the ENDF/B-V and ENDF/B-VI continuous cross-section libraries and S(a,.) scattering data from the ENDF/B­IV file.8 n/y transport was performed using geometry splitting, weight window (WWG) and direct statistical approach (DSA) vari­
9 
ance reduction techniques.8, 
A detailed Mante Carla model of the TRIGA Mark II research reactor has been developed, where ali important details con­cerning the reactor core, graphite reflector, thermal and thermalizing column, irradiation channels and biological shielding were con­sidered (Figure 1). The original TRIGA core 
Radio/ 011co/ 1999; 33(1): 69-75. 

Maucec Metal./ Epithermal 11eutro11 bcm11 for BNCT ni }SI TRI GA renctor 

Figure l. 30 Montc Cmlo model of the JSI TRI GA Mark ll research reactor. 
configuration No. 147 was modified in such a manner that three fuel elements were posi­tioned at the inlet of the radia! channel. With this set-up (based on an MCNP TRIGA benchmark model11 ), which considered 50 fresh fuel elements with multiplication factor keff being 1.0100 ± 0.0017, the total neutron flux was enhanced by 40%, with a negligible change of neutron spectrum.10 
On the basis of an extensive Monte Carlo pilot study of epithermal neutron filter and gamma shielding materials, the fina! configu­ration of the BNCT irradiation facility was elaborated, consisting of the following ele­ments (Figure 2): 12,13 -30 cm Al + 40 cm Al203 (with a density of 
2.3 g/cm3 , which can be achieved by pressing the Al203 powder (initial density is 1.6 g/cm3) at 400-500 kg/cm2) in a sin­gle piece, used as epithermal neutron fil­ter, 

-an additional 30 cm Al0with 0.05 cm of 
23 
Cd foil, used as a thermal neutron absor­

ber, -15 cm Pb, used as a gamma shield and -15 cm Pb + 15 cm borated paraffin (90 vol. 
% C30H62 + 10 vol. % boric acid H3B03), as 
additional neutron/gamma shield. 

Ali the elements are cylindrically shaped and equipped with six small stainless steel wheels to enable unrestrained transport through the channel. In addition to numer­ous benefits offered by this design -easy han­dling and transportation of filter elements, as well as storage after irradiation -it has one major drawback: an approx. 2.5 mm air gap, leading directly from the reactor core to the irradiation point, thus allowing fast neutrons and gamma rays to stream through the gap and contributing significantly to the total dose. When the calculations were repeated using the MC model without the air gap, the fast neutron and gamma doses were reduced by more than 80%! The results of Monte 
Radio/ 011col 1999; 33(1): 69-75. 

Maucec Metal./ Epithermal neutron beam for BNCT at ]SI TRI GA reactor 
watc-r 
Figure 2. MCNP model of the BNCT facility inserted in the radia! channel (units in cm). 
Carla calculations at the irradiation point (Figure 2) are presented in Table 1. 
Experimental verification 
Based on the results of MC calculations, the irradiation facility far BNCT treatment was manufactured. The details of elaboration were presented in more detail.13 The measure­ments of neutron flux and mixed n/y field dosimetry were perfarmed with a set13 of 115In and 197 Au (n,y) activation detectors far the thermal and epithermal range. A Cd cover (thickness 0.5 mm) was used in order to determine the cadmium ratio of the field. The integral fluxes in the fast neutron range were measured with the fallowing threshold reac­tions: 115In(n,n')115min, 27 Al(n,p)27Mg, 56Fe(n,p)56Mn, 64Zn(n,p)64Cu, 24Mg(n,p)24Na, 27 Al(n,a)24Na and 19F(n,2n)18F. Pure metallic fails (In, Al, Fe, Zn and Mg) as well as Teflon™ (CF2) were irradiated with Cd covers. Gamma dose was measured using a set of TLD detectors on the central axis of the chan­nel. In order to absorb the radiation from elec­trons and soft X-rays from the outside and to affirm the reproducibility of the results, the set was confined in a 1 cm thick holder made of plexiglass. To obtain the neutron spectrum (Figure 3), the detector responses were adjust-
Radiol Oncol 1999; 33(1): 69-75. 

MaieCalo --Exl]9imrt 
. . . . . . w w w 
Energy !McVJ 
Figure 3. Calculated vs. measured neutron flux per unit lethargy. 
ed with the SAND-II deconvolution code.14 The systematic error of the experiment was estimated to be less than 3%. The standard deviation over the entire spectrum of detector saturated activities was less than 2%. 
Results 
Experimental results confirmed 10% higher epithermal neutron flux <I> . (0.4 eV < E < 10 
ep, keV) at the irradiation point than MC calcu­lated one. This remains two orders of magni­tude below the recommended therapeutic limit of 109n/cm2s, thus dictating quite long irradiation times. Furthermore, the experiment confirmed a surplus of thermal neutrons (E<0.4 eV) (<I>tennf <I> epi = 1.85 far Mante Carla calcula­tions results and 1.75 far the experiment). This can be attributed to thermal neutrons that arrive at the irradiation point from sur­rounding regions, i.e. the water and concrete of the reactor biological shield (those emerg­ing from the neutron beam itself were cut-off with the 0.5 mm thick Cd absorber). The measured neutron spectrum (Figure 3) con­forms the calculated one; discrepancies emerge only in the fast part of the spectrum (above 10 keV) but stili remain within the 10% confidence interval of the Mante Carla calculated total fast neutron flux. 
<J)nrasn (10 keV<E<300 keV) 

Maucec Me/ a/. / Epit/Jermal 11eutron beam far BNCT at ]SI TRI GA reactor 
Table 1. Results of MC calculations vs. experiment 
Quantityc Method Trerapeutic Monte Carlo" Experimentb limit values 
<l>nlcrm (E<0.4 eV) 6.95e+6 (15) 7.1e+6 (+10) / 
3.75e+6 (12) 4.1e+6 (+20) >109 
3.86e+5 (17) 8.7e+5 (+10)d / 
<I>nra ,,2 (300 keV<E<20 Me V)  4.83e+5 (15)  /  /  
Jepiler  2.55e+6 (12)  /  >5*108  
Dnfast  7.04e+6 (13.8)  7.4e+6 (+15)  /  
Dy  6.07e+7 (11.7)  8.le+7 (+45)  /  
D nfos/<l> ncpitcr  19  18  <5  
Dy/<l>,wpiler  162  197  <3  
Jep/<J)epi  0.68  /  >0.5  

a -() -rclative crrors in% 
b -() -discrepancy from Monte Carlo results in% c _ units: <!>,, and -[n/crn2s], Dfosl and Dy-[10-12 Gy s-'], Dr,,s/<l\,epHer and Dy/<l>n itc, -[10-13 Gy cm2] 
nncp

d -fast neutron flux, measured in a singlc energy group (10 keV<E<20 Me V) 
To determine the influence of the air gap surrounding the filter configuration on the neutron and gamma dose, the profiles of the neutron and gamma fields were measured using photo-luminescence imaging plates (IP) with a 0.1 mm thick dysprosium screen, fre­quently used in the direct method of neutron radiography. The imaging plate is exposed with the neutron-activated Dy foil and later scanned with a laser reader. The gamma beam profile at the outlet of the radia! chan­nel is presented in Figure 4. 
The experimentally measured gamma dose exceeds the calculated one by 33%, thus making the relative gamma dose (Dy/cp epi in units of 10-13 Gy cm2) unacceptably high and almost 60-times higher than therapeutically recommended. The contribution of the 2.5 mm air gap is most clearly evident from Fig­ure 5: the gap region is approximately 30­times more irradiated than the filter-covered region in relative PSL (-1:hoto .S.timulated Luminescence) units as a function of the spa­tial co-ordinate. 


Figure 4. The profile of the gamma profile field at the outlet of the channel (P,eacto, . 2kW, Tirrad 

500s). 
Discussion 

Experimental verification of the BNCT irradia­tion facility at the JSI TRIGA reactor proves the quality and wide applicability of the devel­oped 3-D Mante Carla model, particularly of the reactor core and irradiation channels. The model can easily be extended far the purposes of Prompt Neutron Gamma Activation Analy­sis (PNGAA), Proton Recoil Spectrometry and 
Radio/ Oncol 1999; 33(1): 69-75. 

Maucec Meta/./ Epilhennal 11eu/ron beam far BNCT at ]SI TRIGA reaclor 
many other activities. It is accurate enough to enable agreement with the experimentally obtained results within the confidence inter­vals of the Monte Carlo calculations. 
. 00 
]10 
2 
o 
"' 
o. 
1-.----.-....--... 
o 20 40 m so 100 120 140 
111111 
Figure 5. Numerical profile of the gamma field in relative PSL units (vertical cross-section of Figure 4). 
In the current stage of development, the irradiation facility is not appropriate for clini­cal BNCT treatments. Due to high attenua­tion of the epithermal neutron flux, we were obliged to alter the initial design of the facili­ty so as to move the irradiation point from the outlet of the radia! channel to the interior immediately behind the lead gamma filter. This makes our radiation facility appropriate for in vitro studies of novel techniques of boron entrapment in malignant tumour cells 
(i.e. by application of electroporation 15,16) or in vivo irradiation of smaller laboratory ani­mals. Using stop (or shelter) made of Pb or Bi (gamma shielding) and LiF or LiCO(ther­
23 
mal neutron cut-off), the influence of the air­gap on the irradiation point would be signifi­cantly reduced, thus increasing the quality of the epithermal neutron beam. 
This work represents the first stage of the BNCT research project in Slovenia leading, hopefully, towards further development of a clinical irradiation facility for BNCT treat­ment of human patients in the thermalizing17 
Radio/ Onco/ 1999; 33(1): 69-75. 

or thermal column of the Jožef Stefan Insti­tute TRIGA reactor. 
Acknowledgements 
The authors would like to express special thanks to dr. Kenneth W. Burn from ENEA, Bologna, Italy for extensive help with the variance reduction method DSA for the Monte Carlo calculations, and to dr. Anthony 
R. Byrne, Jožef Stefan Institute, Ljubljana, for a thorough review of this paper. 
References 
1. 
Rubin P. Editor's Note. Int. J Rad 011co/ Biol P/111s 1994; 28: 1055-6. 

2. 
New radiotherapy targets malignant brain tumors, Nuclear News, ANS, 1998, p. 62-3. 

3. 
Despot DN, Kos J, Serša G, Cemažar M, Škrk J, Gubenšek F. Boronated CDI 315B monoclonal antibody and its potential use in baron neutron capture therapy. In Larsson B, Crawfard J, Weinre­ich R, editors. Advances in neutron cap/ure lherapy. Vol. 11, C/zemistry & Biology. Amsterdam: Elsevier; 1997. p. 512-6. 

4. 
Yanagie H, Fujii Y, Tomita T, Sekiguchi M, Eriguchi M, Kobayashi T et al. Tumor growth inhi­bitions in "in-vitro" baron neutron capture reac­tions using liposomal delivery of 10B compound, In Larsson B, Crawfard J, Weinreich R, editors. Adva11ces i11 11e11lro11 cap/ure thcrapy. Vol. II, Chem­islry & Biology. Amsterdam: Elsevier; 1997. p. 491-7. 

5. 
Carlsson J, Edwards K, Ghaneolhosseini H, Gedda L, Johnsson M, Sjoberg S. Stabilised lipo­somes with double targeting intended far use in BNCT, Abstract Book of the Eight International Symposium on Neutron Capture Therapy far Can­cer, Sept 13-18, 1998, La Jolla, CA, USA, p. 184. 

6. 
Rant J, Krištof E, Rudman D, Kavšek D, Maucec M, Glumac B et al. Dosimetry properties oj TRIGA Mark II reactor irradiation fields far tile purposes oj BNCT experiments, Part I, IJS-DP-7634, January 1997 (in Slovene). 

7. 
Serša G, Cemažar M, Novakovic S, Rant J, Krištof E, Miklavcic U et al. Radiation effect on baron neutron capture therapy on mouse melanoma and breast carcinorna cells in vitro. Advances in neulron 



Maucec Met al./ Epitlzemza/ 11eutro11 beam far BNCT at ]SI TRI GA reactor 
cap/ure therapy. Vol. II, Che111istry & Biology. Amster­for BNCT i11 radia/ c/zannel of TR/GA Mark II reactor, 

dam: Elsevier; 1997. p. 480-4. 

8. Briesmeister J, Ed., MCNP -A general 111011/e car/o N-partic/e tra11spart code, Ver. 4B, LA-12625-M, March 1997. 
9. 
Burn KW, A new weight-dependent direct statisti­cal approach model, Nuc/ Sci E11g 1997; 125: 128­70. 

10. 
Maucec M, Glumac B. Developrnent of irradiation facility for BNCT at the JSI TRIGA research reac­tor. In Larsson B, Crawford J, Weinreich R, edi­tors. Adva11ces in 11e11tro11 capture therapy. Vol. /, Medicine & Physiscs. A111sterda111: Elsevier; 1997. p. 370-6. 

11. 
Jeraj R, Glumac B, Maucec M. Monte Carlo simu­lation of the TRIGA Mark II benchmark experi­ment. Nuc/ Tec/mol 1997; 120: 179-87. 


12. 
Maucec M. Mante Car/o ca/cu/ations of 11e11tro11 a11d photon transport in complex geometries, PhD Disser­tation in preparation (in Slovene). 

13. 
Maucec M. Elaboration of epitlzermal ne11tro11 facility 


IJS-DP-7 755, april 1998 (in Slovene) 

14. SAND ll -Neutro11 flux spectra deter111i11atio11 by mul­tiple foil activation . . Oak Ridge National Laborato­ry, USA, Jan 1994. 
15. 
Škrk J, Cemažar M, Mitrovic B, Šorli M, Serša G. Boron entrapment in B16 melanoma cells and turnors by application of electric pulses, Abstract Book of the Eight lnternational Syrnposiurn on Neutron Capture Therapy for Cancer, Sept 13-18, 1998, La Jolla, CA, USA, p. 48. 

16. 
Ono K, Kinashi Y, Masunaga Y, Suzuki M, Takaga­ki M. lncreasc in the effect of 10B-BSH based neu­tron capture therapy by electroporation, Abstract Book of the Eight International Symposium on Neutron Capture Therapy for Cancer, Sept 13-18, 1998, La Jolla, CA, USA, p. 113. 

17. 
Maucec M, Glurnac B. Feasibility of the utilisation of BNCT in the thermalizing column of TRIGA reactor, Abstract Book of the Eight International Symposium on Ncutron Capture Therapy for Can­cer, Sept 13-18, 1998, La Jolla, CA, USA, p. 45. 


Radio/ 011col 1999; 33(1): 69-75. 

Sloveninn abstract 
Radio/ Onco/ 1999; 33(1): 1-8. 
Farmakološko-mehanska tehnika za selektivno trombolizo na perifernem žilju 
Hadjiev J, Horvath L, Mezi'ifi B, Szalay G 
Namen: Pri selektivnem fibrinoliticnem zdravljenju z nizkimi dozami smo nameravali še mehansko izboljšati ucinek unicevanja trombusov z uporabo pulzne razpršilne brizgalke. Predvidevali smo, da borno obenem skrajšali cas zdravljenja in omejili število zapletov po zdravljenju. Metode: V raziskavo smo vkljucili 17 bolnikov z zamašenimi arterijami na spodnjih okoncinah. Zdravili smo jih s selektivno pulzne razpršilno trombolizo. Pri vseh smo upoštevali standardni pro­tokol zdravljenja s trombolizo. S pulzno razpršilno brizgalko Angiodynamics® smo v žilo vbrizgavali streptokinazo -Kabikinase® (5.000-20.000 IU/h), urokinazo -Ukidan® (20.000-40.000 IU/h) in heparin v fiziološki raztopini (500-1.000 IU/h), dokler se trombus ni popolnoma razpustil. Rezultati: Od 1995 do 1997 srno uspešno pozdravili 17 bolnikov. Povprecni cas zdravljenja je bil 12 ur. V tem casu ni bilo hujših zdravstvenih zapletov. Kirurški poseg je bil v prvih šestih mesecih po zdravl­jenju potreben le pri dveh bolnikih in sicer zaradi ponovitve klinicnih simptomov. Zakljucek: Farmakološko-mehanska selektivna trornboliza s pomocjo pulzno-razpršilne tehnike je zanesljiva, sorazmerno hitrejša in tudi varnejša metoda za zagotavljanje prepustnosti zamašenih arterij. 
Radio/ Oncol 1999; 33(1): 9-14. 
Odkrivanje preobremenitve z železom in jeterne ciroze z analizo podatkov o spektralnih znacilnostih CT-ja jeter z visoko locljivostjo 
Kurbel S, Kristek B, Kovacic D, Glavina K, Kurbel B 
Izhodišca. Jeterno cirozo in preobremenitev z železom srno odkrivali z analizo podatkov, dobljenih iz skupka slikanj jeter s CT-jern z visoko locljivostjo. Podatke smo obdelali s statisticnimi metodami in Fourierjevo analizo. Bolniki in metode. Obravnavali smo 11 bolnikov z jeterno cirozo, 7 hernodializiranih bolnikov s preo­bremenjenostjo z železom in 51 posameznikov za primerjavo. Uporabili smo CT Siemens Somatom DHR (kernel 2; 0,2 mm široki, 2 mm debeli piksli). Podrocja vzorcenja so bila kvadratne velikosti, velika 50 pikslov. Vsakemo bolniku srno dolocili tri podrocja posameznikom za primerjavo pa dva. Podrocja smo statisticno obdelali in razgradili v 100 linearnih fragmentov (50 linij in 50 stolpcev s 50 piksli) za Fourierjevo analizo. Za vsak vzorec srno izracunali povprecje in standardno devijacijo. Rezultati. CT visoke locljivosti se je pri bolnikih z obremenitvijo z železom razlikoval od kontrolnih vrednosti po povišani povprecni gostoti (>=79 HU) in zmanjšani moci mnogih harmonij (p<0.01). CT visoke locljivosti pri bolnikih s cirozo pa je pokazal zmanjšano gostoto (<50 HU) in povecano moc harmonij od 0.1 do 0.9 ciklov/mm (valovne dolžine od 10 do 1.1 mm) (p< 0.01). Zakljucki. S pomocjo podatkov, dobljenih s CT-jem jeter z visoko locljivostjo, lahko razlikujemo med normalnimi, ciroticnimi ali z železom preobremenjenimi jetri. 
Radio/ Oncol 1999; 33(1): 77-82. 

S/ovenian abstract 
Radio/ Onco/ 1999; 33(1): 15-21. 

Standardizirana slikovna dokumentacija v nuklearni medicini 
Bohuslavizki KH, Buchert R, Mester J, Clausen M 
V vsakdanji medicinski praksi ni splošno sprejetih standardov o slikovni dokumentaciji. Tako nastanejo težave pri ponovnem ocenjevanju slikovnih zapisov na rentgenskih filmih ali papirju, ki jih dobimo iz drugih nuklearnomedicinskih ustanov ali pa je težavna primerjava razlicnih slikovnih zapisov pri istem bolniku. Da bi olajšali ponovno ocenjevanje slikovnih zapisov, smo izdelali predloge za potrebno dokumentacijo. Opisani so primeri slikovnega dokumentiranja pri najbolj pogostih nuklearnomedicinskih raziskavah, kot so scintigrafija pljuc, šcitnice, kosti -s planarno ali SPECT kamero, pa tudi funkcionalna ledvicna scintigrafija, perfuzijska miokardna scintigrafija in pozitronska emisijska scintigrafija. Ti primeri imajo namen vzpodbuditi razpravo v nuleranomedicinski stroki, kakšni naj bodo slikovni dokumentacijski zapisi pri nuk­learnomedicinskih preiskavah. 
Radio/ Onco/ 1999; 33(1): 23-6. 

Odkrivanje metastaz osteogenega sarkoma v limfnih bezgavkah z 99mTc-MDP scintigrafijo. Prikaz primera 
IlicD, Zafirovski G, Simova N, Zisovski N, Glamocanin S, Janevska V, Tolevska C, Vaskova O, Samardziski M 
Osteogeni sarkom se po telesu obicajno širi hematogeno. Tako nastanejo pljucne in skeletne metastaze. Redkeje pa se širi limfogeno. Prikazujemo primer bolnika z osteogenim sarkomom, pri katerem smo z radioizotopno scintigrafijo kosti odkrili metastaze v limfnih bezgavkah. 

Radio/ Oncol 1999; 33(1): 77-82. 

S/uve11ia11 abstract 
Radio/ Oncol 1999; 33(1): 27-33. 
Korelacija med NK citoliticnim in BLT esteraznim testom pri dolocevanju aktivnosti NK celis stimuliranih s tumorskimi celicami 
IhanA 
Z meritvami encimske aktivnosti N-benzyloxycarboxy-L-lizinske esteraze srno preiskovali eksoc­itozo celic NK. Celice NK smo osamili iz levkocitnega koncentrata (Buffy coat) oz. venske krvi. Eksocitozo smo sprožili s kombinacijo PMA/ionomcin ali s tumorskimi celicami K542. Ob stim­ulaciji celic NK s tumorskimi celicami (K542) smo opazovali znacilno soodvisnost (Cor=0.84) med rezultati citoliticne aktivnosti celic NK in med izmerjenimi encimskimi aktivnosti N-benzy­loxycarboxy-L-lizinske esteraze (BLT test). Ugotavljamo, da BLT test omogoca nadrobnejši študij dogodkov, ki se odvijajo med citotoksicno aktivnostjo celic NK. 
Radio/ Oncol 1999; 33(1): 35-41. 
Mikro jedra v limfocitih z zavrto citokinezo pri bolnikih po radioterapiji z I-131 
Kašuba V, Kusic Z, Garaj-Vrhovac V 
Pri 10 bolnikih s hipertireozo in razlicnimi vrstami karcinoma šcitnice smo raziskovali mikro jedra (MN) v limfocitih periferne krvi, v katerih citohalazin-B zavira citokinezo. Bolniki so bili zdravljeni z I-131 natrijevim jodidom, ki so ga dobili peroralno, v odmerkih 259-5180 MBq. Frekvenco v mikro jedrih smo izmerili pred zdravljenjem z I-131 in po njem. Rezultate merjenja pred zdravljenjem smo uporabili kot kontrolo. Rezultati naše raziskave so bili glede na starost in trenutno dejavnost precej variabilni. Iz števila mikro jeder in dvojedrnih celic z mikro jedri (BNMN) ter s pomocjo Poissonove regresije, prirerejene autokorelaciji za vsak osebek posebej, je bilo mogoce ugotavljati pretirano disperzijo. Samo pri zacetni dozi nismo ugotovili znacilne pomembnosti. Pri medsebojnem vplivanju casa in odmerka, še zlasti pri višjih odmerkih, pa smo ugotovili znacilno pomebnost, medtem ko so spremembe v jedrih potekale pocasneje. Za najnižji odmerek (259 MBq) smo izracunali relativni cas tveganja. Ce smo odmerek podvojili, se je število mikro jeder in dvojedrnih celic z mikro jedri zmanjšalo povprecno za 5% na dan (rela­tivno tveganje MN = 0.955; relativno tveganje 

0.954). 

BNMN 
Radio/ Onco/ 1999; 33(1): 77-82. 

Slovenian abstmct 
Radio/ Onco/ 1999; 33(1): 43-53. 

Napovedni pomen urokinaznega aktivatorja plazminogena in njegovih inhibitorjev pri bolnicah z rakom dojk 
Borštnar S, Cufer T, Vrhovec I 
Urokinazni aktivator plazminogena (u-PA), in njegova inhibitorja (PAI-1 in PAI-2) igrajo pomembno vlogo pri razgradnji medcelicnega tkiva in s tem pri prodoru tumorskih celic v okoli­co ter metastaziranju. Namen naše raziskave je bil ugotoviti morebitno napovedno vrednost uPA, PAI-1 in PAI-2 v retrospektivni seriji 87 bolnic z rakom dojke stadijev I-III, njihove citosole hranimo na Onkološkem inštitutu v Ljubljani. Srednja opazovalna doba je bila 35 mesecev. Napovedni pomen uveljavljenih napovednih dejavnikov ter uPA, PAI-1 in PAI-2 smo ocenjevali z univariatno statisticno analizo in delnimi multivariatnimi modeli. Vrednost uPA so bile zelo nizke in niso korelirale s preživetjem brez znamenj bolezni, PAI-1 in PAI-2 pa sta znacilno vpli­vala na cas prve ponovitve bolezni. Bolnice, ki so imele vrednost PAI-1 vecjo od 5nglmg pro­teinov, so imele statisticno znacilno slabše preživetje brez znamenj bolezni kot bolnice z manjšimi vrednostmi (58% vs 85% , p = 0.0046). PAI-2 je pokazal nasprotno sliko, bolnice z vred­nostmi PAI-2 vecjimi od 6.4 nglmg proteinov so imele statisticno znacilno boljše triletno preživetje brez znamenj bolezni kot bolnice z manjšimi vrednostmi ( 90% vs 48%, p = 0.0178). PAI-1 in PAI-2 sta ohranila svojo neodvisno napovedno vrednost ob dodajanju uveljavljenih napovednih dejavnikov v delne multivariatne modele in le lokalna razširjenost bolezni je pokazala vecjo napovedno moc od PAI-1 in PAI-2. 
Radio/ Oncol 1999; 33(1): 55-8. 

Radioterapija neovaskularizirane žilnice pri starostni makularni degeneraciji 
Wagner W, Beeke E, Barsnick P 
Starostna makularna degeneracija zaradi neovaskularizacije žilnice predstavlja v oftalmologiji vse vecji problem. Zdravljenje doslej ni bilo prevec uspešno in obenem povezano s številnimi stranskimi ucinki. Pred kratkim pa smo zasledili ugodne rezultate zdravljenja z obsevanje z nizkimi dozami. Zato smo ponovno proucili podatke o bolnikih, vkljucenih v pilotsko študijo, da bi lahko potrdili v literaturi objavljene podatke. Raziskava je zajemala 43 bolnikov, ki smo jih obsevali z lineranim akceleratorjem (6 MV) pri skupni dozi 16Gy. Šest mesecev po zakljucenem zdravljenju se je pri 69% bolnikov ostrina vida ohranila ali celo izboljšala. Pri proucevanju stran­skih ucinkov pa v casu spremljanja bolezni v povprecju 12 mesecev nismo zasledili niti akutnih niti kasnih posledic zdravljenja. 

Radio/ Onco/ 1999; 33(1): 77-82. 

5/ovenian a/Jstracl 
Radio/ Onco/ 1999; 33(1): 59-61. 


Sekundarni malignomi po zdravljenju seminomov z radioterapijo 
Fuchshuber PR, Lee RJ, McGrath B, Gibbs JF, Kraybill WG, Proulx GM 
Opisujemo primer bolnika, ki je bil pred 19-imi leti adjuvantno zdravljen z radioterapijo zaradi seminoma, stadij I, nato pa je zbolel zaradi malignega perifernega tumorja živcnih ovojnic, ki je nastal v obsevalnem polju. Razpravljamo o novejših podatkih, ki govore o morebitnem tveganju bolnikov s seminomom, da bi zboleli za sekundarnim malignomom glede na ostalo populacijo. Tudi opisani primer bolnika kaže na potrebo skrbne evaluacije dosedanje radioterapije pri bol­nikih z zgodnjo obliko seminoma. 
Radio/ Oncol 1999; 33(1): 63-8. 


Sarkom mehurja: prikaz primera s pregledom literature 
Proulx GM, Gibbs JF, Lee RJ, Velagapudi S, Huben R 
Sarkomi mehurja so redki tumorji, zato temeljita prognoza bolnika in odlocitev o vrsti zdravljen­ja na redkih porocilih posamicnih ustanov. Tako tudi nimamo standardne razvrstitve bolnikov po stadijih bolezni, niti ne standardnega zdravljenja. Na Roswell Park inštitutu v Buffalu smo naredili retrospektivni pregled bolnikov, ki so se od jan­uarja 1960 do decembra 1997 zdravili zaradi sarkoma mehurja. Te izkušnje in pregled literature nam omogocajo boljše razumevanje naravnega poteka bolezni in napovedi bolnikovega preživet­ja glede na vrste zdravljenja. V clanku prikazujemo primer bolnika, ki smo ga radikalno zdravili z namenom ozdravitve in podajamo izkušnje drugih ustanov. Ce je bolezen omejena na mehur, lahko bolnika zdravimo s popolno ali delno kirurško odstran­itvijo mehurja ali pa se odlocimo za kombinirano zdravljenje z delno odstranitvijo mehurja, obsevanjem in kemoterapijo, saj s tem zmanjšamo nevarnost sistemskega obolenja in in ima bolnik dobre možnosti, da ga pozdravimo. 
Radio/ Oncol 1999; 33(1): 77-82. 

5/ovenian abstract 
Radio/ 011co/ 1999; 33(1): 69-75. 

Epitermicni izvor nevtronov za terapijo raka z zajetjem nevtronov v boru na reaktorju TRIGA Instituta Jožef Stefan -modeliranje in eksperimentalna preveritev 
Maucec M, Glumac B, Rant J, Krištof E 



Zaradi specificnih lastnosti obratovanja spadajo reaktorji TRIGA med najvarnejše raziskovalne reaktorje. V svetu se jih vedno vec uporablja kot izvor termicnih oz. epitermicnih nevtronov v namene terapije raka z zajetjem nevtronov v boru (angl. ]3.oron Neutron .apture Iherapy). Prispevek prikazuje rezultate Monte Carlo modeliranja, optimizacije ter eksperimentalne veri­fikacije obsevalne naprave z epitermicnimi nevtroni razvite v radialnem kanalu reaktorja TRIGA na Institutu Jožef Stefan v Ljubljani. Rezultati potrjujejo široko uporabnost razvitega tri-dimen­zionalnega modela reaktorja, predvsem sredice in obsevalnih kanalov. Zaradi visoke atenuacije nevtronskega fluksa (<Pi = 4.lxl06 n/cm2s, kar je dobra dva reda velikosti pod priporoceno ter­
ep apevtsko mejo) obsevalna naprava v obstojeci izvedbi ni primerna za klinicna BNCT obsevanja. Prispevek zracne reže, ki obdaja sistem filtrov dodatno prispeva k visoki specificni dozi gama žarkov in hitrih nevtronov na obsevalnem mestu, ki sta skoraj 60-krat višji od priporocenih vrednosti. Vendar pa bi z minimalno predelavo obsevalne naprave ter uporabo zašcitnega ohišja s Pb (ali Bi) ter LiF (ali Li2CO3) lahko obcutno izboljšali kvaliteto obsevalnega curka kot orodja za "in-vitro" študije transporta bora v malignih celicnih kulturah ali laboratorijskih živalih. 
Radio/ Onco/ 1999; 33(1): 77-82. 

Radio/ 011col 1999; 33(1): 83-5. 
Notices 
Notices submitted for publication should co11t11i1111 mailing address, phone and/or fax number and/or e-mažl oj a contact person or department. 
Radiotherapy 

Marc/z 1-5, 1999 
A Practical and theoretical Course in Radiotherapy Physic. Part B: Brachytherapy, Radiobiology and Treatment machines. 
Contact Dr Alan Nahum, tel: +44 '181 64260, ext 3309; or fax +44 181 6433 812; or e-mail alan«vicr .ac.uk 
Thoracic surgery 

Marc/z 4-5,1999 
IV international Mecting on General Thoracic Surgery will be held in Barcelona, Spain. 
Contact Congress Secretariat, RCT, Aulestia i Pijoan, 12, Baixos, E-088012 Barcelona; or call +34 93 415 6938; or fax: +34 93 415 6904 
Radiation oncology 

Marc/z 4-6,1999 
Third Annual Meeting of the Scientific Association Swiss Radiation Oncology, SASRO, PSI will be held in Villingen, Switzerland. 
Contact Secr. Radiation Medicine; tel: +41 56 3103524, or fax +41 56 3103515; or c-mail radiation.medicine.1>psi.ch 
Lung cancer 

March 4-6,1999 
First World Conference on clinical co-opcrativc Research far Lung Cancer will take place in Brussels, Belgium. 


Contact European Lung Cancer Working Party c/o Prof. J-P. Sculier, Institute Jules Bordet, 1, rue 1-leger­Bordet, B1000, Brussel, Belgium, or call +32 2 539 04 96; or fax +32 2 534 37 56; or e-mail 1(l1473.1044«Dcompuserve.com 
As a service to our readers, 110/ices oj 111eelings ar courses wi/1 /Je i11serted fi-ee oj clznrge. Please send i11jor111atio11 to lhe Edilorial ojjice, Radiology and Oncology, Vrazov trg 4, 1105 Ljubljana, Slovenia. 

Head and neck cancer 

Marc/z 10-12, 1999 The ESO training course will take place in Cairo, Egypt. 
Contact ESO office far Balkans and Middle East, N. Pavlidis, E. Andreopoulou Medica! School, Depart­ment of Medica] Oncology, University 1-lospital of loannina, 45110 loannina, Greecc; or call +30 651 99394 or +30 953 91083; or fax +30 651 97505 
Radiation oncology 

March 10-14, 1999 
Course on Evidence-Based Radiation Oncology: Principles and Methods, will take place in Cape Town, South Africa. 
Contact ISRO Secretariat, av Mounier 83/12, 1200 Brussels, Belgium, or call +32 2 7759342, or fax +32 2 7795494; or e-mail ISRO«2estro.be 
Radiation oncology 

Marc/z 14-18, 1999 Course on Modern Brachytherapy Tcchniques will bc held in Oslo, Norway. 
Contact ESTRO office, Av. E. Mounierlaan, 83/4, B­1200 Brussels, Bclgium; or call + 32 7759340; or fax +32 2 7795494; or e-mail infoQ,>estro.be; web: http://www.estro.be 
Lung cancer 

March 19-21,1999 
Thc ESO training course will take place in Zakopane, Poland. Contact ESO office for Central and Eastern Europe, Ms. Dagmar Just, Arztckammer fo Wien, Fortbil­
4th 

dungsreferat, Weihburggasse 10-12, floor, 1010 Vienna, Austria; or call +43 1 51501262; or fax +43 1 51501200; or e-mail just«1>aekwicn.or.at 
84 Notices 
Breast cancer 
March 22-25, 1999 
First Acta Oncologica Symposium on Management of the axilla in Breast cancer. lmplications far Diagno­sis, Prognosis, Treatment and Morbidity will take place in Geilo, Norway. 
Contact Jens Overgaard, Experimental Clinical Oncology Department, Aarhus University 1--Iospital, Norrebrogade, 44, Building 5, 8000 Aarhus C, Den­mark, or call +45 89 49 26 29, or fax: +45 86 19 71 09 
Radiation treatment planing 
March 24-27, 1999 
Principles and Practice of 3-D Radiation Treatment Planning will be held in Munich, Germany. Contact DR. 1--IJ Feldmann, Radiooncology Depart­ment, Technische Universitiit Mi.inchen, Klinikum rechts der Isar, or call +4 49 8941404512/11, or fax +49 8941404587 
Radiophysics 
April 6-11, 1999 
5th Biennial ESTRO Meeting on Physics far Clinical Radiotherapy will be held in Gottingen, Germany. 
Contact ESTRO office, Av. E. Mounierlaan, 83/4, B­1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail infa@estro.be; web: http://www.estro.be 
New drugs in cancer treatment 
April 12-14,1999 
The ESO training course will take place in Smolenice Castle, Slovakia. Contact ESO office far Central and Eastern Europe, Ms. Dagmar Just, Arztekammer fii Wien, Fortbil­
4th 
dungsreferat, Weihburggasse 10-12, floor, 1010 Vienna, Austria; or call +43 1 51501262; or fax +43 1 51501200; or e-mail just@aekwien.or.at 
Radiotherapy 
April 12-15, 1999 
1--Iadron Beam Therapy, Physics and Biology, will take place in Cape Town, South Africa. 
Contact Dr Dan Jones, National Accelerator Centre, P O. Box 72, Faure, 7131, South Africa, or call +27 21 834 3820, or fax: +27 21 834 3382 
Radio/ Onco/ 1999; 33(1): 83-5. 


Clinical research 
April 18-22, 1999 
ESTRO course on Methodology of Clinical Research, will be held in Como, ltaly. 
Contact ESTRO office, Av. E. Mounierlaan, 83/4, B­1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail infa@lestro.be; web: http://www.estro.be 
Colorectal cancer 
May, 1999 
The ESO training course will take place in Nicosia, Cyprus. 
Contact ESO office far Balkans and Middle East, N. Pavlidis, E. Andreopoulou Medica] School, Depart­ment of Medica] Oncology, University 1--Iospital of Ioannina, 45110 Ioannina, Greece; or call +30 651 99394 or +30 953 91083; or fax +30 651 97505 
Good clinical practice in oncology 
May 6-7, 1999 
The ESO training course Good Clinical Practice Scientific Aspects: What Kind of Clinical Trials Do Need in Oncology will take place in Vienna, Austria. 
Contact ESO office far Central and Eastern Europe, Ms. Dagmar Just, Arztekammer fii Wien, Fortbil­dungsreferat, Weihburggasse 10-12, 4th floor, 1010 Vienna, Austria; or call +43 1 51501262; or fax +43 1 51501200; or e-mail just@aekwien.or.at 
Gynecological oncology 
May 8-13, 1999 
The 11 th lnternational Meeting of Gynecological Oncology will take place in Budapest, 1--lungary. Contact Prof. Dr. Peter B6sze, 1301 Budapest, PO. Box 46, 1--Iungary; or fax + 36 1 275 2172 
Brachitherapy 
May 10-12, 1999 
Annual Brachytherapy Meeting GEC-ESTRO, will take place in Utrecht, Netherlands. 
Contact ESTRO office, Av. E. Mounierlaan, 83/4, B­1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail infa@estro.be; web: http://www.estro.be 

Notices 85 
Breast pathology -oncology 

May 11-15, 1999 The ESO training course will take place in loanni­na, Greece. 
Contact ESO office for Balkans and Middle East, N. Pavlidis, E. Andreopoulou Medica! School, Depart­ment of Medica! Oncology, University Hospital of Ioannina, 45110 Ioannina, Greece; or call +30 651 99394 or +30 953 91083; or fax +30 651 97505 
Radiology 

May 17-19, 1999 The UK's Premier Radiological Meeting will be held in Birmingham, United Kingdom. 
Contact Radiology 1999 Secretariat, 36 Portland Place, London, WIN 4AT, UK; or call + 44 (O) 171 307 1410; or fax + 44 (O) 171 307 1414 
Paediatric oncology 

May 17-19, 1999 
The training course under the auspices of the Inter­national Society of Paediatric Oncology will be held in Amsterdam, the Netherlands. 
Contact P.A. Voete, call +31 20 5665655; or fax +31 20 6912231 
Euthanasia in oncology 

May 21-23, 1999 The ESO training course will take place in loanni­na, Greece. 
Contact ESO office for Balkans and Middle East, N. Pavlidis, E. Andreopoulou Medica! School, Depart­ment of Medica! Oncology, University Hospital of Ioannina, 45110 Ioannina, Greece; or call +30 651 99394 or +30 953 91083; or fax +30 651 97505 
Paediatric oncology 

May 24-29, 1999 
The training course under the auspices of the Inter­national Society of Paediatric Oncology will be held in Moscow, Russia. 

Contact P.A. Voete, call +31 20 5665655; or fax +31 20 6912231 
Epidemiology in cancer 

May 27-29, 1999 The ESO training course will take place in Olo­mouc, Czech Republic. Contact ESO office for Central and Eastern Europe, Ms. Dagmar Just, Arztekammer fii Wien, Fortbil­
dungsreferat, Weihburggasse 10-12, 4th floor, 1010 Vienna, Austria; or call +43 1 51501262; or fax +43 1 51501200; or e-mail just@aekwien.or.at 
Lymphoma 

May 28 -June l, 1999 The Postgraduate ESMO/ESO course will take place in Mante Verit4, Ascona, Switzerland. 
Contact the ESMO Head office, Via Soldino 22, 6900 Lugano, Switzerland; or call +41 91 950 07 86; or fax + 41 91 959 07 87; or e-mail esmosecr@dial.eunet.ch 
Molecular oncology 

May 30 -June 3, 1999 Course on Molecular Oncology for Radiotherapy will be held in lzmir, Turkey. 
Contact ESTRO office, Av. E. Mounierlaan, 83/4, B­1200 Brussels, Belgium; or call +32 7759340; or fax +32 2 7795494; or e-mail info@estro.be; web: http://www.estro.be 
Paediatric and medica! oncology 

May 31 -June 3, 1999 
The ESO training course "Basic Science and Treat­ment in Paediatric and Medica! Oncology" will take place in Yerevan. 
Contact ESO Office for Russia artd Community of Independent States, M. Vukelic, CSC Ltd, Heiligen­stadter Strasse, 395b, 1190 Vienna, Austria; or call +43 1 3690444; or fax +43 1 369044420 
Lymphoma 

June 2-5, 1999 The VII. lnternational Conference on Malignant Lymphoma will be held in Lugano, Switzerland. 
Contact the ESMO Secretariat, Via Soldino 22, 6900 Lugano, Switzerland; or call +41 91 967 54 11; or fax +41 91 967 57 44 
High <lose chemotherapy and autologous stem cell transplantation 

June 3-5,1999 
The ESO training course High Dose Chemotherapy and Autologous Stem Celi Transplantation in Cancer: Indications and Unresolved Questions will take place in Brijuni Island. 
Contact ESO office for Central and Eastern Europe, Ms. Dagmar Just, Arztekammer fii Wien, Fortbil­dungsreferat, Weihburggasse 10-12, 4th floor, 1010 Vienna, Austria; or call +43 1 51501262; or fax +43 1 51501200; or e-mail just@aekwien.or.at 
Radio/ Onco/ 1999; 33(1): 83-5. 


FONDACIJA "DOCENT DR. J. CHOLEWA" JE NEPROFITNO, NEINSTITUCIONALNO IN NESTRANKARSKO ZDRUŽENJE POSAMEZNIKOV, USTANOV IN ORGANIZACIJ, KI ŽELIJO MATERIALNO SPODBUJATI IN POGLABLJATI RAZISKOVALNO DEJAVNOST V ONKOLOGIJI. 
MESESNELOVA 9 61000 LJUBLJANA TEL 061 1 5 91 277 FAKS 061 21 81 13 
ŽR: 50100-620-133-05-10331 15-214779 

Activity of "Dr. J. Cholewa" foundation for cancer research and education -report for the first quarter of 1999 
In the first quarter of 1999 the "Dr. J. Cholewa" foundation far cancer research and edu­cation continued with its activities, as already outlined in previous reports. The activity of the Foundation is slowly gathering momentum, and it is thus necessary to acknowledge the efforts of the majority of its members and supporters. As the activity of the Foundation needs considerable financial support, additional efforts shall be required to guarantee its continuation at the present leve!. Worldwide financial crisis left its impact on the willing­ness of the donors to contribute to the Foundation, which nevertheless managed to collect enough support not to compromise its previously stated goals. 
Despite the problems mentioned in the previous paragraph, the Foundation continues to support regular publication of "Radiology and Oncology" international scientific journal, and the regular publication of the "Challenge ESO Newsletter", the newsletter of the Euro­pean School of Oncology from Milan, Italy. Both medica! journals mentioned are edited and published in Ljubljana, Slovenia. It is also worth mentioningthe educational grants awarded to three oncologists for their training in foreign countries, as well as the support the Foundation granted to the Organizing Committee of the 1st. Slovenian Congress of Surgery, held in the city of Maribor. Ali of this in addition to the grants and support men­tioned before in the previous reports in "Radiology and Oncology" journal. 
For 1999 the Foundation plans to continue to help with the provision of grants for the various European School of Oncology courses, and to support publishing and editorial activity frorn the various fields of oncology. Additional administrative help will thus be needed to ensure proper and smoothe execution of the Foundation's goals, with the neces­sary steps already taken in this direction. 
" Dr. J. Cholewa" Foundation for Cancer Research and Education continues to pursue its goals, as defincd by its statute and meetings of the Board of directors in the past year. It continues to cooperate with sirnilar institutions in Slovenia and abroad, especially with the "Mali Vitez" Foundation from Ljubljana, Slovenia. In the rest of 1999 additional steps will have to be taken to further consolidate the Foundation financially. 
Tomaž Benulic, MD Borut Štabuc, MD, PhD Andrej Plesnicar, MD 


INTERNATIONAL INC. 

DISTRIBUTED BY 
SIRION s.r.1. 
34170 GORIZIA-ITALY Corso Italia 112 Tel. (0481) 32073-32074 Fax.(0481)534753 
varno in ucinkovito lajšanje bolecin, tudi ponoci 

RETARD 
. lx l)m,, /4M/ 

Sestava: V lakirani tableti je 100 mg tramadol hidroklorida. Oprema: Tramal retard 100 mg -škatlica s 30 lakiranimi tabletami. Izdeluje: Bayer Pharma d.o.o. Ljubljana po licenci GrGnenthal GmbH 
BayerEB 
GRUNENTHAL 


I:SANOLABOR 
,,.., c::=J ,,,,,-_ 
.C::,Q.Q 

Pri nas dobite vse za rentgen! 
• KODAK • SIEMENS • GENERAL ELECTRIC • • PHILIPS • BENNETT • HITACHI • POLAROID • • XENOLITE • MAVIG • CAWO • 
• 
rentgenski filmi in kemikalije 

• 
kontrastna sredstva 


• 
rentgenska zašcitna sredstva 

• 
rentgenski aparati, aparati za ultrazvocno diagnostiko, stroji za avtomatsko razvijanje, negatoskopi in druga oprema za rentgen 


!: 511.NOLII.BOR, Leskoškova 4, 1103 Ljubljana Tel.: 06 l l 85 42 l l Fax: 06 l 140 l 3 04 

Nycomed lmaging is proud of its role in providing for the early, accurate diagnosis of disease, thus improving patients' quality of life and prospect for effective treatment. The company is commited to the continuous development of inovative imaging product to enhance 
diagnostic procedures. 
ZASTOPA DISTRIBUCIJA HIGIEA d.o.o., Trzin Nycomed SALUS d.d. Ljubljana 
tel.: (061) 1897 225 tel.: (061) 1899 100 
Amersham 
fax: (061) 1897 226 fax: (06L) 1681420 
GEMZAR® 
gemcitabin hidroklorid 



Nova Uic v 
onkologiji 

INDIKACIJE 
• 
nemikrocelicni pljucni karcinom (NSCLC) 

• 
karcinom pankreasa 

• 
karcinom secnega mehurja 



Drugi terapevtski ucinki opaženi pri karcinomih dojke, ovarijev, prostate in pri mikrocelicnem pljucnem karcinomu (SCLC). 
Oblika in pakiranje 
• 
injekcijska steklenicka z 200 mg gemcitabina 

• 
injekcijska steklenicka z 1 g gemcitabina 



Sestavine aktivna ucinkovina (gemcitabin hidroklorid), manitol, natrijev acetat, natrijev hidroklorid 
Dodatne informacije o zdravilu so na voljo v strokovnih publikacijah, ki jih dobite na našem naslovu. 
Eli Lilly (Suisse) S. A., Podružnica v Ljubljani, 
Ljubljana, Vošnjakova 2, tel.: (061) 319-648, faks: (061) 319-767 
r11..1 
ONKOLOGIJA 
KNOWLEDGE IS POWERFUL MEDICINE 
SIE ENS Rešitve po meri 

Mammomat 3000 modular 
Mammomat 3000 modular 
e univerzalni sistem za vse vrste mamografije • optimizacija doze in kompresije z OPDOSE in OPCOMP sistema e modularna zgradba zagotavlja posodabljanje sistema 
• servis v Sloveniji z zagotovljenimi rezervnimi deli in garancijo e izobraževanje za uporabnike 
SIEMENS d.o.o. Dunajska 22 1511 Ljubljana Telefon 061/1746 100 Telefaks 061/1746 135 

.@B@ULJWB[Q) 
U,4)./JAJ r:i . .. 
LABORMED d.o.o. . SL0-1215 Med.vode . Zg. PirniLe 96/c Telefon 061 / 621 098 . Telefax 061 / 621 415 
Labormed v Sloveniji ekskluzivno zastopa naslednje firme: 
KOTTERMANN 
laboratorijsko pohištvo, varnostne omare za kisline, luge, topila, pline in strupe, ventilacijska tehnika in digestoriji 
EHRET 
laminar flow tehnika, inkubato,ji, sušilniki. suhi ster­ilizatorji in oprema za laboratorijsko vzrejo živali -kletke 
DAKO 
testi za aplikacijo v imunohistokemiji, patologiji, mikrobiologiji, virologiji, 1110110-in poliklonalna pro­titelesa ... 
GFL 
laboratorijski aparati, omare in skrinje za globoko zatnrzovanjc 
ROSYS ANTHOS 
fotometri, avtomatski pralni sistem za mikrotitrine plošce 
CHARLES ISCHI Pharma-Pri.iftechnik 
specialna oprema za testiranje izdelkov v farmacevt­ski industriji; aparati za procesno kontrolo in kontro­lo kvalitete 
NOVODIRECT BIOBLOCK 
kompletna oprema in pripomoaki za delo v labora­toriju 
ANGELANTONI SCIENTIFICA 
hladilna tehnika in aparati za laboratorije, transfuzi­ologijo, patologijo in sodno medicino 
INTEGRA BIOSCIENCES 
laboratorijska oprema za mikrobiologijo, biologijo celic, molekularno biologijo in biotehnologijo 
CORNINGCostar 
specialna laboratorijska plastika za aplikacijo v imunologiji, mikrobiologiji, virologiji, ipd., mehan­ske eno-in veckanalne pipete ter nastavki 
BIOMERICA 
hitri testi za diagnostiko, EIA / RIA testi 
Advanced BIOTECHNOLOGIES 
PCR tehnologija, potrošni material, reagenti, encimi 
SVANOVA Biotech 
Elisa testi za diagnostiko v veterini 
HURNER 
ventilacijska tehnika 


Are your radiographers' hands ticd by cquipmcnt thar slows rhem down? And what can you do abour it? Nearly 70% of X-rays are made at a Bucky. A facr rhar has influenced rhe design of Philips' Bucky systems. lt's led to technology withour gadgetry, for uncomplicated, speedy work. Control units operated one-banded. 
A display that eliminares trips to the generator. Tomography functions acrivated via a single button. Ali to speed patient throughput, for improved cost-cfficicncy. Tt's one way in which Philips Medica! Systems is working with you to meet roday's changing hcalrhcare needs. Far more information call 061 177 88 50. 
website: -.phllips.com/ms 
-

-
-
-
-
111111 
v svetu najvec predpisovani sistemski( antimikotik 
edini peroralni sistemski antimikotik za zdravljenje vaginalne kandidoze, ki ga je odobril FDA 
Skrajšano navodilo Flukonazol je sistemski antimikotik iz skupine triazolov. 
Odmerjanje pri razlicnih indikacijah: 
vaginalna kandidoza 150 mg v enkratnem odmerku 
mukozna kandidoza 50 do 100 mg na dan 
dermatomikoze 50 mg na dan ali 150 mg na teden 
sistemska kandidoza prvi dan 400 mg, nato od 200.do 400 mg na dan Najvecji dnevni odmerek je 800 mg. 
oreorecevanie kandidoze 50 do 400 ma na dan 
kriptokokni meningitis prvi dan 400 mg, nato od 200 do 400 mg na dan 
Kontraindikacije: Preobcutljivost za zdravilo ali sestavine odmerku flukonazola za zdravljenje vaginalne kandidoze . Pri veckratnih in vecjih odmerkih so možne interakcije s terfenadinom, cisaprld9111c varfarlnom, derivati sulfonilureje, hidroklorotiazidom, fe,nitoinom. rifa"mniohiom. teofilinom, indinavirom in midazolamom. Nosecnost in le, ce je korist zdravljenja za mater vecja od tveganja za s flukonazolom ne dojijo. Stranski ucinki: Povezani so napenjanje, bolecine v trebuhu, driska, zelo redko se po1avIJo. preo1:JautJJivostI anafilaksija in angioedem -v tem primeru takoj prenehamo jemati z.d, glivicnimi obolenji lahko pride do levkopenije in tromhncitooeniie in do encimov. Oprema in nacin izdajanja: 7 150 mg. Na zdravniški recept 1/99. Podrobnejše informacije so na voljo pri proizvajalcu, 

Lekarne, bolnišnice, zdravstveni domovi in veterinarske ustanove vecino svojih nakupov opravijo pri nas. Uspeh našega poslovanja temelji na kakovostni ponudbi, ki pokriva vsa podrocja humane medicine in veterine, pa tudi na hitrem in natancnem odzivu na zahteve naših kupcev. 
KEMOFARMACIJA-VAŠ ZANESLJIVI DOBAVITELJ! 
KEMOFARMACIJA 
Veletrgovina za oskrbo zdravstva, d.d. / l 000 Ljubljana, Cesta na Brdo l 00 Telefon: 061 12-32-145 / Telefax: 271-5B8, 271-362 
E\ecsys, Reaching New Heights 

Roche and Boehringer Mannheim, two of the most experienced innovators in diagnostics, have joined forces to create a new, fully­integrated approach to health management worldwide. 
There are no limits to innovation at Roche Diagnostics. 
Roche Diagnostics Parmova 53 tel.:* 386 61 133 60 24 
Info Office Ljubljana 1000 Ljubljana *386611336331 Slovenia fax: * 386 61 130 92 02 
Radiology 1111d Oncology 


Instructions for authors 
Editorial policy of the journal Radiology and Oncology is to publish original scientific pa­pers, professional papers, review articles, case reports and varia (editorials, reviews, short communications, professional information, book reviews, letters, etc.) pertinent to diag­nostic and interventional radiology, computer­ized tornography, magnetic resonance, ultra­sound, nuclear medicine, radiotherapy, clini­cal and experimental oncology, radiobiology, radiophysics and radiation protection. The Editorial Board requires that the paper has not been published or submitted for publication elsewhere: the authors are responsible for all staternents in their papers. Accepted articles become the property of the journal and there­fore cannot be published elsewhere without written perrnission from the editorial board. Papers concerning the work on hurnans, must comply with the principles of the declaration of Helsinki (1964). The approval of the ethical cornmittee must then be stated on the manu­script. Papers with questionable justification will be rejected. 
Manuscript written in English should be submitted to the Editorial Office in triplicate (the original and two copies), including the illustrations: Radiology and Oncology, Institute of Oncology, Vrazov trg 4, SI-1000 Ljubljana, Slovenia; (Phone: +386 61 132 00 68, Tel./Fax: +386 61 133 74 10, E-mail: gsersa@onko-i.si). Authors are also asked to submit their manu­scripts on a 3.5" 1.44 Mb formatted diskette. The type of computer and word-processing package should be specified (Word for Win­dows is preferred). 
Ali articles are subjected to editorial review 

and review by independent referee selected by 
the editorial board. Manuscripts which do not 
comply with the technical requirements stated 
herein will be returned to the authors for cor­rection before peer-review. Rejected manu­scripts are generally returned to authors, how­ever, the journal cannot be held responsible for their loss. The editorial board reserves the right to ask authors to make appropriate changes in the contents as well as grammati­cal and stylistic corrections when necessary. The expenses of additional editorial work and requests for reprints will be charged to the authors. 
General instructions• Radiology and Onco­logy will consider manuscripts prepared according to the Vancouver Agreement (N Engl J Med 1991; 324: 424-8, BMJ 1991; 302: 6772; JAMA 1997; 277: 927-34.). Type the manu­script double spaced on one side with a 4 cm margin at the top and left hand side of the sheet. Write the paper in grammatically and stylistically correct language. Avoid abbrevia­tions unless previously explained. The techni­cal data should conforrn to the SI system. The rnanuscript, including the references may not exceed 15 typewritten pages, and the number of figures and tables is limited to 4. If appro­priate, organize the text so that it includes: Introduction, Material and methods, Results and Discussion. Exceptionally, the results and discussion can be cornbined in a single sec­tion. Start each section on a new page, and number each page consecutively with Arabic numerals. 


Title page should include a concise and infonnative title, followed by the full name(s) of the author(s); the institutional affiliation of each author; the name and address of the cor­responding author (including telephone, fax and e-mail), and an abbreviated title. This should be followed by the abstract page, sum­rnarising in less than 200 words the reasons 
for the study, experimental approach, the major findings (with specific data if possible), and the principal conclusions, and providing 3­6 key words for indexing purposes. Structured abstracts are preferred. If possible, the authors are requested to submit also slovenian version of the title and abstract. The text of the report should then proceed as follows: 
Introduction should state the purpose of the article and summarize the rationale for the study or observation, citing only the essential references and stating the aim of the study. 
Material and methods should provide enough information to enable experiments to be repeated. New methods should be described in detail. Reports on human and animal subjects should include a statement that ethical approval of the study was obtained. 
Results should be presented clearly and concisely without repeating the data in the tables and figures. Emphasis should be on clear and precise presentation of results and their significance in relation to the aim of the investigation. 
Discussion should explain the results rather than simply repeating them and interpret their significance and draw conclusions. It should review the results of the study in the light of previously published work. 
Illustrations and tables must be numbered and referred to in the text, with appropriate location indicated in the text margin. Illustra­tions must be labelled on the back with the author's name, figure number and orienta­tion, and should be accompanied by a descriptive legend on a separate page. Line drawings should be supplied in a form suit­able for high-quality reproduction. Pho­tographs should be glossy prints of high qual­ity with as much contrast as the subject allows. They should be cropped as close as possible to the area of interest. In pho­tographs mask the identities of the patients. Tables should be typed double spaced, with descriptive title and, if appropriate, units of numerical measurements included in column heading. 
References must be numbered in the order in which they appear in the text and their cor­responding numbers quoted in the text. Authors are responsible for the accuracy of their references. References to the Abstracts and Letters to the Editor must be identified as such. Citation of papers in preparation, or submitted for publication, unpublished obser­vations, and personal communications should not be included in the reference list. If essen­tial, such material may be incorporated in the appropriate place in the text. References fol­low the style of Index Medicus. All authors should be listed when their number does not exceed six; when there are seven or more authors, the first six listed are followed by "et al.". The following are some examples of refer­ences from articles, books and book chapters: 
Dent RAC, Cole P. In vitro maturation of monocytes in squamous carcinoma of the lung. Br J Cancer 1981; 43: 486-95. 
Chapman S, Nakielny R. A guide to radiolog­ical procedures. London: Bailliere Tindall; 1986. 
Evans R, Alexander P. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. Immunobiology oj macrophage. New York: Acad­emic Press; 1976. p. 45-74. 
Page proofs will be faxed to the corre­sponding author whenever possible. It is their responsibility to check the proofs carefully and fax a list of essential corrections to the editorial office within 48 hours of receipt. If corrections are not received by the stated deadline, proof-reading will be carried out by the editors. 
Reprints: Fifty reprints are free of charge, for more contact editorial board. 
Far reprint infonnation in North America Contact: International Reprint Corporation 968 Admiral Callaghan Lane,# 268 P.O. Box 12004, Vallejo; CA 94590, Tei: (707) 553 92 30, Fax: (707) 552 95 24. 


Za mirno 
potovanje skozi kemoterapijo 
N . 
avouan 
troptsteron 

• 
preprecevanje slabosti in bruhanja pri emetogeni kemoterapiji 

• 
ucinkovito zdravilo, ki ga odrasli in otroci dobro prenašajo 

• 
vedno 1-krat na dan 

• 
vedno 5 mg 




Skrajšano navodilo za uporabo: Navoban® kapsule, Navoban® raztopina za injiciranje 2 mg in S mg. Serotoninski antagonist. Oblika in sestava: 
1 trda kapsula vsebuIe S mg tropJSetronovega hidroklorida. 1 ampula po 2 ml vsebu1e 2 mg tropJSetronovega hidroklorida. 1 ampula po S ml vsebuje 
S mg tropisetronovega hidroklorida. Indikacije: Preprecevanje slabosti In bruhanja, ki sta posledici zdravljen1a s citostatiki. Zdravljenje pooperativne slabosti in bruhanja. Preprecevanje pooperat,vne slabosti in bruhanja pri bolnicah, pn katerih je nacrtovana ginekološka operacija v trebušni votlini. Odmerjanje in uporaba: Preprecevanje slabosti in bruhanja, ki sta posledic, zdravljenja s citostatiki. Odmerjanje pri otrocih: Otroci starejši od 2 let 0,2 mg/kg telesne mase na dan. Najvecji dnevni odmerek ne sme preseci S mg. Prvi dan kot intravenska infuzija ali kot pocasna intravenska injekcija. Od 2. do 6. dne naj otrok Jemlje zdravilo oralno (raztopino v ampuli razredcimo s pomarancnim sokom ali koka kolo). Odmerjanje pri odraslih: 6-dnevna kura po S mg na dan. Prvi dan kot intravenska infuzija ali pocasna intravenska injekcija. Od 2. do 6. dne 1 kapsula na dan. Zdravljenje in preprecevanje pooperativne slabosti in bruhanja: Odmerjanje pri odraslih: 2 mg Navobana z intravensko ,nfuziJO ali kot pocasno injekcijo. GleJ celotno navodilo! Kontraindikacije: Preobcutljivost za tropisetron, druge antagoniste receptorjev 5-HT3 ali katerokoli sestavino zdravila. Navobana ne 
smemo dajati nosecnicam; izJema je preprecevanje pooperat,vne slabost, in bruhanja pri kirurških posegih, katerih del je tudi terapevtska prekinitev nosecnosti. Previdnostni ukrepi: Bolniki z nenadzorovano hipertenzijo; bolniki s prevodnimi ali drugimi motnjami srcnega ritma; ženske, ki dojijo; bolnik,, ki upravljajo s stroji ali vozili. Medsebojno delovanje zdravil: R1fampicin ali druga zdravila, ki induciraJo Jetrne encime. GleJ celotno navodilo! Stranski ucinki: Glavobol, zaprtje, redkeje omotica, utruienost in prebavne motnje (bolecine v trebuhu in driska), preobcutljivostne reakcije. Zelo redko kolaps, sinkopa ali zastoJ srca, vendar vzrocna zveza z Navobanom ni bila dokazana. Nacin izdajanja: Kapsule: uporaba samo v bolnišnicah, izjemoma se izdaja na zdravniški recept pri nadaljevanju zdravljenja na domu ob odpustu iz bolnišnice in nadaljnJem zdravljenju. Ampule: uporaba samo v bolnišnicah. Oprema in odlocba: Zloženka s S kapsulami po S mg; številka odlocbe 512/8-773/97 z dne 10.11.1997. Zloženka z 1 ampulo po 2 ml (2 mg/2 ml); številka odlocbe 512/8-772/97 z dne 10. 11. 1997. Zloženka z 10 ampulami po S ml (S mg/S ml); številka odlocbe 512/8-771/97 z dne 
1 O. 11. 1997. Izdelovalec: NOVARTIS PHARMA AG, Basel, Švica. Imetnik dovoljenja za promet z zdravilom: NOVARTIS PHARMA SERVICES INC., Podružnica v Sloveniji, Dunajska 22, 1511 Ljubljana, kjer so na voljo informacije in literatura. 
Preden predpišete Navoban, prosimo preberite celotno navodilo. 
' 

NOVARTIS